Intercellular adhesion molecules and their binding ligands

ABSTRACT

The present invention relates to intercellular adhesion molecules (ICAM-1) which are involved in the process through which lymphocytes recognize and migrate to sites of inflammation as well as attach to cellular substrates during inflammation. The invention is directed toward such molecules, screening assays for identifying such molecules and antibodies capable of binding such molecules. The invention also includes uses for adhesion molecules and for the antibodies that are capable of binding them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/474,388, filed on Jun. 7, 1995, pending; which is a divisional ofU.S. application Ser. No. 08/186,456, filed Jan. 25, 1994, now U.S. Pat.No. 5,612,216, which is a divisional of U.S. application Ser. No.07/515,478, filed Apr. 27, 1990, now U.S. Pat. No. 5,284,931; said U.S.application Ser. No. 08/186,456, is a continuation-in-part of U.S.patent application Ser. No. 07/045,963, filed on May 4, 1987, nowabandoned, 07/115,798, filed on Nov. 2, 1987, now abandoned, 07/155,943,filed on Feb. 16, 1988, now abandoned, 07/189,815, filed on May 3, 1988,now abandoned, 07/250,446, filed on Sep. 28, 1988, now abandoned,07/324,481, filed on Mar. 16, 1989, now abandoned, 07/373,882, filed onJun. 30, 1989, now abandoned, and 07/456,647, filed on Dec. 22, 1989,now abandoned, all of which applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to intercellular adhesion molecules suchas ICAM-1 which are involved in the process through which populations oflymphocytes recognize and adhere to cellular substrates so that they maymigrate to sites of inflammation and interact with cells duringinflammatory reactions. The present invention additionally relates toligand molecules capable of binding to such intercellular adhesionmolecules, to a screening assay for these ligands, and to uses for theintercellular adhesion molecule, the ligand molecules, and the screeningassay.

2. Description of the Related Art

Leukocytes must be able to attach to cellular substrates in order toproperly defend the host against foreign invaders such as bacteria orviruses. An excellent review of the defense system is provided by Eisen,H. W., (In: Microbiology, 3rd Ed., Harper & Row, Philadelphia, Pa.(1980), pp. 290-295 and 381-418). They must be able to attach toendothelial cells so that they can migrate from circulation to sites ofongoing inflammation. Furthermore, they must attach toantigen-presenting cells so that a normal specific immune response canoccur, and finally, they must attach to appropriate target cells so thatlysis of virally-infected or tumor cells can occur.

Recently, leukocyte surface molecules involved in mediating suchattachments were identified using hybridoma technology. Briefly,monoclonal antibodies directed against human T-cells (Davignon, D. etal., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)) and mouse spleencells (Springer, T. et al. Eur. J. Immunol. 9:301-306 (1979)) wereidentified which bound to leukocyte surfaces and inhibited theattachment related functions described above (Springer, T. et al., Fed.Proc. 44:2660-2663 (1985)). The molecules identified by those antibodieswere called Mac-1 and Lymphocyte Function-associated Antigen-1 (LFA-1).Mac-1 is a heterodimer found on macrophages, granulocytes and largegranular lymphocytes. LFA-1 is a heterodimer found on most lymphocytes(Springer, T. A., et al. Immunol. Rev. 68:111-135 (1982)). These twomolecules, plus a third molecule, p150, 95 (which has a tissuedistribution similar to Mac-1) play a role in cellular adhesion (Keizer,G. et al., Eur. J. Immunol. 15:1142-1147 (1985)).

The above-described leukocyte molecules were found to be members of arelated family of glycoproteins (Sanchez-Madrid, F. et al., J. Exper.Med. 158:1785-1803 (1983); Keizer, G. D. et al., Eur. J. Immunol.15:1142-1147 (1985)). This glycoprotein family is composed ofheterodimers having one alpha chain and one beta chain. Although thealpha chain of each of the antigens differed from one another, the betachain was found to be highly conserved (Sanchez-Madrid, F. et al., J.Exper. Med. 158:1785-1803 (1983)). The beta chain of the glycoproteinfamily (sometimes referred to as “CD18”) was found to have a molecularweight of 95 kd whereas the alpha chains were found to vary from 150 kdto 180 kd (Springer, T., Fed. Proc. 44:2660-2663 (1985)). Although thealpha subunits of the membrane proteins do not share the extensivehomology shared by the beta subunits, close analysis of the alphasubunits of the glycoproteins has revealed that there are substantialsimilarities between them. Reviews of the similarities between the alphaand beta subunits of the LFA-1 related glycoproteins are provided bySanchez-Madrid, F. et al., (J. Exper. Med. 158:586-602 (1983); J. Exper.Med. 158:1785-1803 (1983)).

A group of individuals has been identified who are unable to expressnormal amounts of any member of this adhesion protein family on theirleukocyte cell surface (Anderson, D. C., et al., Fed. Proc. 44:2671-2677(1985); Anderson, D. C., et al., J. Infect. Dis. 152:668-689 (1985)).Lymphocytes from these patients displayed in vitro defects similar tonormal counterparts whose LFA-1 family of molecules had been antagonizedby antibodies. Furthermore, these individuals were unable to mount anormal immune response due to an inability of their cells to adhere tocellular substrates (Anderson, D. C., et al., Fed. Proc. 44:2671-2677(1985); Anderson, D. C., et al., J. Infect. Dis. 152:668-689 (1985)).These data show that immune reactions are mitigated when lymphocytes areunable to adhere in a normal fashion due to the lack of functionaladhesion molecules of the LFA-1 family.

Thus, in summary, the ability of lymphocytes to maintain the health andviability of an animal requires that they be capable of adhering toother cells (such as endothelial cells). This adherence has been foundto require cell-cell contacts which involve specific receptor moleculespresent on the cell surface of the lymphocytes. These receptors enable alymphocyte to adhere to other lymphocytes or to endothelial, and othernon-vascular cells. The cell surface receptor molecules have been foundto be highly related to one another. Humans whose lymphocytes lack thesecell surface receptor molecules exhibit chronic and recurringinfections, as well as other clinical symptoms including defectiveantibody responses.

Since lymphocyte adhesion is involved in the process through whichforeign tissue is identified and rejected, an understanding of thisprocess is of significant value in the fields of organ transplantation,tissue grafting, allergy and oncology.

SUMMARY OF THE INVENTION

The present invention relates to Intercellular Adhesion Molecule-1(ICAM-1) as well as to its functional derivatives. The inventionadditionally pertains to antibodies and fragments of antibodies capableof inhibiting the function of ICAM-1, and to other inhibitors of ICAM-1function; and to assays capable of identifying such inhibitors. Theinvention additionally includes diagnostic and therapeutic uses for allof the above-described molecules.

In detail, the invention includes the intercellular adhesion moleculeICAM-1 or its functional derivatives, which are substantially free ofnatural contaminants. The invention further pertains to such moleculeswhich are additionally capable of binding to a molecule present on thesurface of a lymphocyte.

The invention further pertains to the intercellular adhesion moleculeICAM-1, and its derivatives which are detectably labeled.

The invention additionally includes a recombinant DNA molecule capableof expressing ICAM-1 or a functional derivative thereof.

The invention also includes a method for recovering ICAM-1 insubstantially pure form which comprises the steps:

(a) solubilizing ICAM-1 from the membranes of cells expressing ICAM-1,to form a solubilized ICAM-1 preparation,

(b) introducing the solubilized ICAM-1 preparation to an affinitymatrix, the matrix containing antibody capable of binding to ICAM-1,

(c) permitting the ICAM-1 to bind to the antibody of the affinitymatrix,

(d) removing from the matrix any compound incapable of binding to theantibody and

(e) recovering the ICAM-1 in substantially pure form by eluting theICAM-1 from the matrix.

The invention additionally includes an antibody capable of binding to amolecule selected from the group consisting of ICAM-1 and a functionalderivative of ICAM-1. The invention also includes a hybridoma cellcapable of producing such an antibody.

The invention further includes a hybridoma cell capable of producing themonoclonal antibody R6-5-D6.

The invention further includes a method for producing a desiredhybridoma cell that produces an antibody which is capable of binding toICAM-1, which comprises:

(a) immunizing an animal with a cell expressing ICAM-1,

(b) fusing the spleen cells of the animal with a myeloma cell line,

(c) permitting the fused spleen and myeloma cells to form antibodysecreting hybridoma cells, and

(d) screening the hybridoma cells for the desired hybridoma cell that iscapable of producing an antibody capable of binding to ICAM-1.

The invention includes as well the hybridoma cell, and the antibodyproduced by the hybridoma cell, obtained by the above method.

The invention is also directed to a method of identifying anon-immunoglobulin antagonist of intercellular adhesion which comprises:

(a) incubating a non-immunoglobulin agent capable of being an antagonistof intercellular adhesion with a lymphocyte preparation, the lymphocytepreparation containing a plurality of cells capable of aggregating;

(b) examining the lymphocyte preparation to determine whether thepresence of the agent inhibits the aggregation of the cells of thelymphocyte preparation; wherein inhibition of the aggregation identifiesthe agent as an antagonist of intercellular adhesion.

The invention is also directed toward a method for treating inflammationresulting from a response of the specific defense system in a mammaliansubject which comprises providing to a subject in need of such treatmentan amount of an anti-inflammatory agent sufficient to suppress theinflammation; wherein the anti-inflammatory agent is selected from thegroup consisting of: an antibody capable of binding to ICAM-1; afragment of an antibody, the fragment being capable of binding toICAM-1; ICAM-1; a functional derivative of ICAM-1; and anon-immunoglobulin antagonist of ICAM-1.

The invention further includes the above-described method of treatinginflammation wherein the non-immunoglobulin antagonist of ICAM-1 is anon-immunoglobulin antagonist of ICAM-1 other than LFA-1.

The invention is also directed to a method of suppressing the metastasisof a hematopoietic tumor cell, the cell requiring a functional member ofthe LFA-1 family for migration, which method comprises providing to apatient in need of such treatment an amount of an anti-inflammatoryagent sufficient to suppress the metastasis; wherein theanti-inflammatory agent is selected from the group consisting of: anantibody capable of binding to ICAM-1; a fragment of an antibody, thefragment being capable of binding to ICAM-1; ICAM-1; ICAM-1; afunctional derivative of ICAM-1; and a non-immunoglobulin antagonist ofICAM-1.

The invention further includes the above-described method of suppressingthe metastasis of a hematopoietic tumor cell, wherein thenon-immunoglobulin antagonist of ICAM-1 is a non-immunoglobulinantagonist of ICAM-1 other than LFA-1.

The invention also includes a method of suppressing the growth of anICAM-1-expressing tumor cell which comprises providing to a patient inneed of such treatment an amount of a toxin sufficient to suppress thegrowth, the toxin being selected from the group consisting of atoxin-derivatized antibody capable of binding to ICAM-1; atoxin-derivatized fragment of an antibody, the fragment being capable ofbinding to ICAM-1; a toxin-derivatized member of the LFA-1 family ofmolecules; and a toxin-derivatized functional derivative of a member ofthe LFA-1 family of molecules.

The invention is also directed to a method of suppressing the growth ofan LFA-1-expressing tumor cell which comprises providing to a patient inneed of such treatment an amount of toxin sufficient to suppress suchgrowth, the toxin being selected from the group consisting of atoxin-derivatized ICAM-1; and a toxin-derivatized functional derivativeof ICAM-1.

The invention is further directed toward a method of diagnosing thepresence and location of an inflammation resulting from a response ofthe specific defense system in a mammalian subject suspected of havingthe inflammation which comprises:

(a) administering to the subject a composition containing a detectablylabeled binding ligand capable of identifying a cell which expressesICAM-1, and

(b) detecting the binding ligand.

The invention additionally provides a method of diagnosing the presenceand location of an inflammation resulting from a response of thespecific defense system in a mammalian subject suspected of having theinflammation which comprises:

(a) incubating a sample of tissue of the subject with a compositioncontaining a detectably labeled binding ligand capable of identifying acell which expresses ICAM-1, and

(b) detecting the binding ligand.

The invention also pertains to a method of diagnosing the presence andlocation of an ICAM-1-expressing tumor cell in a mammalian subjectsuspected of having such a cell, which comprises:

(a) administering to the subject a composition containing a detectablylabeled binding ligand capable of binding to ICAM-1, the ligand beingselected from the group consisting of an antibody and a fragment of anantibody, the fragment being capable of binding to ICAM-1, and

(b) detecting the binding ligand.

The invention also pertains to a method of diagnosing the presence andlocation of an ICAM-1-expressing tumor cell in a mammalian subjectsuspected of having such a cell, which comprises:

(a) incubating a sample of tissue of the subject with a compositioncontaining a detectably labeled binding ligand capable of bindingICAM-1, the ligand being selected from group consisting of antibody anda fragment of an antibody, the fragment being capable of binding toICAM-1, and

(b) detecting the binding ligand.

The invention also pertains to a method of diagnosing the presence andlocation of a tumor cell which expresses a member of the LFA-1 family ofmolecules in a subject suspected of having such a cell, which comprises:

(a) administering to the subject a composition containing a detectablylabeled binding ligand capable of binding to a member of the LFA-1family of molecules, the ligand being selected from the group consistingof ICAM-1 and a functional derivative of ICAM-1, and

(b) detecting the binding ligand.

The invention also pertains to a method of diagnosing the presence andlocation of a tumor cell which expresses a member of the LFA-1 family ofmolecules in a subject suspected of having such a cell, which comprises:

(a) incubating a sample of tissue of the subject in the presence of adetectably labeled binding ligand capable of binding to a member of theLFA-1 family of molecules, the ligand being selected from the groupconsisting of ICAM-1 and a functional derivative of ICAM-1, and

(b) detecting the binding ligand which is bound to a member of the LFA-1family of molecules present in the sample of tissue.

The invention additionally includes a pharmaceutical compositioncomprising:

(a) an anti-inflammatory agent selected from the group consisting of: anantibody capable of binding to ICAM-1; a fragment of an antibody, thefragment being capable of binding to ICAM-1; ICAM-1; a functionalderivative of ICAM-1; and a non-immunoglobulin antagonist of ICAM-1, and

(b) at least one immunosuppressive agent selected from the groupconsisting of: dexamethasone, azathioprine and cyclosporin A.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows in diagrammatic form the adhesion between a normal and anLFA-1 deficient cell.

FIG. 2 shows in diagrammatic form the process of normal/normal celladhesion.

FIG. 3 shows the kinetics of cellular aggregation in the absence (X) orpresence of 50 ng/ml of PMS (0).

FIG. 4 shows coaggregation between LFA-1- and LFA-1⁺ cells.Carboxyfluorescein diacetate labeled EBV-transformed cells (104) asdesignated in the figure were mixed with 10⁵ unlabeled autologous cells(solid bars) or JY cells (open bars) in the presence of PMA. After 1.5 hthe labeled cells, in aggregates or free, were enumerated using thequalitative assay of Example 2. The percentage of labeled cells inaggregates is shown. One representative experiment of two is shown.

FIG. 5 shows the immunoprecipitation of ICAM-1 and LFA-1 from JY cells.Triton X-100 lysates of JY cells (lanes 1 and 2) or control lysis buffer(lanes 3 and 4) were immunoprecipitated with antibody capable of bindingto ICAM-1 (lanes 1 and 3) or antibodies capable of binding to LFA-1(lanes 2 and 4). Panel A shows results under reducing conditions; PanelB shows results obtained under non-reducing conditions. Molecular weightstandards were run in lane S.

FIG. 6 shows the kinetics of IL-1 and gamma interferon effects on ICAM-1expression on human dermal fibroblasts. Human dermal fibroblasts weregrown to a density of 8×10⁴ cells/0.32 cm² well. IL-1 (10 U/ml, closedcircles) or recombinant gamma interferon (10 U/ml, open squares) wasadded, and at the indicated time, the assay was cooled to 4° C. and anindirect binding assay was performed. The standard deviation did notexceed 10%.

FIG. 7 shows the concentration dependence of IL-1 and gamma interferoneffects on ICAM-1. Human dermal fibroblasts were grown to a density of8×10⁴ cells/0.32 cm²/well. IL-2 (open circle), recombinant human IL-1(open square), recombinant mouse IL-1 (solid square), recombinant humangamma interferon (solid circles), and recombinant beta interferon (opentriangle) were added at the indicated dilution and were incubated for 4hours (IL-1) or 16 hours (beta and gamma interferon). The indicatedresults are the means from quadruplicate determinations; standarddeviation did not exceed 10%.

FIG. 8 shows the nucleotide and amino acid sequence of ICAM-1 cDNA. Thefirst ATG is at position 58. Translated sequences corresponding toICAM-1 tryptic peptides are underlined. The hydrophobic putative signalpeptide and transmembrane sequences have a bold underline. N-linkedglycosylation sites are boxed. The polyadenylation signal AATAAA atposition 2976 is over-lined. The sequence shown is for the HL-60 cDNAclone. The endothelial cell cDNA was sequenced over most of its lengthand showed only minor differences.

FIG. 9 shows the ICAM-1 homologous domains and relationship to theimmunoglobulin supergene family. (A) Alignment of 5 homologous domains(D1-5). Two or more identical residues which aligned are boxed. Residuesconserved 2 or more times in NCAM domains, as well as resides conservedin domains of the sets C2 and C1 were aligned with the ICAM-1 internalrepeats. The location of the predicted β-strands in the ICAM-1 domain ismarked with bars and lower case letters above the alignments, and theknown location of β-strands in immunoglobulin C domains is marked withbars and capital letters below the alignment. The position of theputative disulfide bridge within ICAM-1 domains is indicated by S—S.(B-D) Alignment of protein domains homologous to ICAM-1 domains;proteins were initially aligned by searching NBRF databases using theFASTP program. The protein sequences are MAG, NCAM, T cell receptor αsubunit V domain, IgMμ chain and α-1-B-glycoprotein.

FIG. 10 shows a diagrammatic comparison of the secondary structures ofICAM-1 and MAG.

FIG. 11 shows LFA-1-positive EBV-transformed B-lymphoblastoid cellsbinding to ICAM-1 in planar membranes.

FIG. 12 shows LFA-1-positive T lymphoblasts and T lymphoma cells bind toICAM-1 in plastic-bound vesicles.

FIG. 13 shows the inhibition of binding of JY B-lymphoblastoid cellbinding to ICAM-1 in plastic-bound vesicles by pretreatment of cells orvesicles with monoclonal antibodies.

FIG. 14 shows the effect of temperature on binding of T-lymphoblasts toICAM-1 in plastic-bound vesicles.

FIG. 15 shows divalent cation requirements for binding of T-lymphoblaststo ICAM-1 in plastic-bound vesicles.

FIG. 16 shows the effect of anti-adhesion antibodies on the ability ofperipheral blood mononuclear cells to proliferate in response to therecognition of the T-cell associated antigen OKT3. “OKT3” indicates theaddition of antigen.

FIG. 17 shows the effect of anti-adhesion antibodies on the ability ofperipheral blood mononuclear cells to proliferate in response to therecognition of the non-specific T-cell mitogen, concanavalin A. “CONA”indicates the addition of concanavalin A.

FIG. 18 shows the effect of anti-adhesion antibodies on the ability ofperipheral blood mononuclear cells to proliferate in response to therecognition of the keyhole limpet hemocyanin antigen. The addition ofkeyhole limpet hemocyanin to the cells is indicated by “KLH.”

FIG. 19 shows the effect of anti-adhesion antibodies on the ability ofperipheral blood mononuclear cells to proliferate in response to therecognition of the tetanus toxoid antigen. The addition of tetanustoxoid antigen to the cells is indicated by “AGN.”

FIG. 20 shows the alignment of ICAM amino-terminal domains.

FIG. 21 shows an ICAM-1 schematic with position of domain deletions.

FIG. 22 shows the expression of ICAM-1 deletion mutants in COS cells.COS cells were analyzed by flow cytofluorometry following indirectimmunofluorescence with RR1/1.

FIG. 23 shows the expression of ICAM-1 deletion mutants in COS cells byflow cytofluorometry following indirect immunofluorescence with MAbsRR1/1 (solid bar), R6.5 (open bar), LB-2 (stippled bar) or CL203(hatched bar). Specific fluorescence intensity was determined withbackground binding to mock transfected cells subtracted.

FIG. 24 shows binding of ICAM-1 deletion mutants to LFA-1 and HRV14. COScells expressing ICAM-1 deletion mutants were tested for adherence toplastic bound LFA-1 and for binding 35S met-labeled HRV14. Standarderror for multiple experiments (2-4) are indicated.

FIG. 25 shows binding of HRV14 to ICAM-1 in the absence of divalentcations. Binding of 35SHRV14 to increasing concentrations of plasticbound ICAM-1 occurred in HRV-buffer with 10 mM Mg++ (open circles) orHRV-buffer minus added Mg++ but with 5 mMEDTA (open triangles). SKW3binding was in HRV buffer with 0.5 mMMg++ (solid circles) or 5 mM EDTA(solid triangles).

FIG. 26 shows a model of ICAM-1 D1 and D2 tertiary structure:Localization of LFA-1 and HRV binding sites. The basic tertiarystructure of an Ig constant domain (Wright, S. D., et al., Proc. Natl.Acad. Sci. USA 85:7734-7738 (1988)) was modified to accommodate thepredicted b strands (wide arrows) and b turns of ICAM-1 D1 and D2(Staunton, D. E., et al., Nature 339:61-64 (1989a)). Residues involvedin LFA-1 or HRV14 binding are indicated. The effect of theircorresponding mutations on LFA-1/HRV14 binding (X-fold decrease) isindicated, respectively (outline print). The position of D2 N-linkedoligosaccharides (open triangle) are indicated.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention relates to the discovery of anatural binding ligand to LFA-1. Molecules such as those of LFA-1family, which are involved in the process of cellular adhesion arereferred to as “adhesion molecules.”

The natural binding ligand of the present invention is designated“Intercellular Aadhesion Molecule-1” or “ICAM-1.” ICAM-1 is a 76-97 Kdglycoprotein. ICAM-1 is not a heterodimer. The present invention isdirected toward ICAM-1 and its “functional derivatives.” A “functionalderivative” of ICAM-1 is a compound which posesses a biological activity(either functional or structural) that is substantially similar to abiological activity of ICAM-1. The term “functional derivatives”¹ isintended to include the “fragments,” “variants,” “analogs,” or “chemicalderivatives” of a molecule. A “fragment” of a molecule such as ICAM-1,is meant to refer to any polypeptide subset of the molecule. Fragmentsof ICAM-1 which have ICAM-1 activity and which are soluble (i.e. notmembrane bound) are especially preferred. A “variant” of a molecule suchas ICAM-1 is meant to refer to a molecule substantially similar instructure and function to either the entire molecule, or to a fragmentthereof. A molecule is said to be “substantially similar” to anothermolecule if both molecules have substantially similar structures or ifboth molecules possess a similar biological activity. Thus, providedthat two molecules possess a similar activity, they are consideredvariants as that term is used herein even if the structure of one of themolecules not found in the other, or if the sequence of amino acidresidues is not identical. An “analog” of a molecule such as ICAM-1 ismeant to refer to a molecule substantially similar in function to eitherthe entire molecule or to a fragment thereof. As used herein, a moleculeis said to be a “chemical derivative” of another molecule when itcontains additional chemical moieties not normally a part of themolecule. Such moieties may improve the molecule's solubility,absorption, biological half life, etc. The moieties may alternativelydecrease the toxicity of the molecule, eliminate or attenuate anyundesirable side effect of the molecule, etc. Moieties capable ofmediating such effects are disclosed in Remington's PharmaceuticalSciences (1980). “Toxin-derivatized” molecules constitute a specialclass of “chemical derivatives.” A “toxin-derivatized” molecule is amolecule (such as ICAM-1 or an antibody) which contains a toxin moiety.The binding of such a molecule to a cell brings the toxin moiety intoclose proximity with the cell and thereby promotes cell death. Anysuitable toxin moiety may be employed; however, it is preferable toemploy toxins such as, for example, the ricin toxin, the diphtheriatoxin, radioisotopic toxins, membrane-channel-forming toxins, etc.Procedures for coupling such moieties to a molecule are well known inthe art.

A “peptidomimetic” of ICAM-1 is a functional derivative of ICAM-1 whosetertiary structure is substantially similar to the tertiary structure ofICAM-1.

An antigenic molecule such as ICAM-1, or members of the LFA-1 family ofmolecules are naturally expressed on the surfaces of lymphocytes. Thus,the introduction of such cells into an appropriate animal, as byintraperitoneal injection, etc., will result in the production ofantibodies capable of binding to ICAM-1 or members of the LFA-1 familyof molecules. If desired, the serum of such an animal may be removed andused as a source of polyclonal antibodies capable of binding thesemolecules. It is, however, preferable to remove splenocytes from suchanimals, to fuse such spleen cells with a myeloma cell line and topermit such fusion cells to form a hybridoma cell which secretesmonoclonal antibodies capable of binding ICAM-1 or members of the LFA-1family of molecules.

The hybridoma cells, obtained in the manner described above may bescreened by a variety of methods to identify desired hybridoma cellsthat secrete antibody capable of binding either to ICAM-1 or to membersof the LFA-1 family of molecules. In a preferred screening assay, suchmolecules are identified by their ability to inhibit the aggregation ofEpstein-Barr virus-transformed cells. Antibodies capable of inhibitingsuch aggregation are then further screened to determine whether theyinhibit such aggregation by binding to ICAM-1, or to a member of theLFA-1 family of molecules. Any means capable of distinguishing ICAM-1from the LFA-1 family of molecules may be employed in such a screen.Thus, for example, the antigen bound by the antibody may be analyzed asby immunoprecipitation and polyacrylamide gel electrophoresis. If thebound antigen is a member of the LFA-1 family of molecules then theimmunoprecipitated antigen will be found to be a dimer, whereas if thebound antigen is ICAM-1a single molecular weight species will have beenimmunoprecipitated. Alternatively, it is possible to distinguish betweenthose antibodies which bind to members of the LFA-1 family of moleculesfrom those which bind ICAM-1 by screening for the ability of theantibody to bind to cells such as granulocytes, which express LFA-1, butnot ICAM-1. The ability of an antibody (known to inhibit cellularaggregation) to bind to granulocytes indicates that the antibody iscapable of binding LFA-1. The absence of such binding is indicative ofan antibody capable of recognizing ICAM-1. The ability of an antibody tobind to a cell such as a granulocyte may be detected by means commonlyemployed by those of ordinary skill. Such means include immunoassays,cellular agglutination, filter binding studies, antibody precipitation,etc.

The anti-aggregation antibodies of the present invention mayalternatively be identified by measuring their ability to differentiallybind to cells which express ICAM-1 (such as activated endothelialcells), and their inability to bind to cells which fail to expressICAM-1. As will be readily appreciated by those of ordinary skill, theabove assays may be modified, or performed in a different sequentialorder to provide a variety of potential screening assays, each of whichis capable of identifying and discriminating between antibodies capableof binding to ICAM-1 versus members of the LFA-1 family of molecules.

In addition to the above-described functional derivatives of ICAM-1,other agents which may be used in accordance of the present invention inthe treatment of inflammation include antibody to ICAM-1, and receptormolecules (such as LFA-1, p150,95, Mac-1, etc.), or fragments of suchmolecules, which are capable of binding to ICAM-1. Since molecules ofthe CD-18 family are able to bind to ICAM-1, administration of suchmolecules (for example as heterodimers having both alpha and betasubunits, or as molecules composed of only an alpha, or a beta subunit,or as molecules having fragments of either or both subunits) is able tocompete with (or exclude) HRV for binding to ICAM-1 present onendothelial cells.

The anti-inflammatory agents of the present invention may be obtained bynatural processes (such as, for example, by inducing an animal, plant,fungi, bacteria, etc., to produce a non-immunoglobulin antagonist ofICAM-1, or by inducing an animal to produce polyclonal antibodiescapable of binding to ICAM-1); by synthetic methods (such as, forexample, by using the Merrifield method for synthesizing polypeptides tosynthesize ICAM-1, functional derivatives of ICAM-1, or proteinantagonists of ICAM-1 (either immunoglobulin or non-immunoglobulin)); byhybridoma technology (such as, for example, to produce monoclonalantibodies capable of binding to ICAM-1); or by recombinant technology(such as, for example, to produce the anti-inflammatory agents of thepresent invention in diverse hosts (i.e., yeast, bacteria, fungi,cultured mammalian cells, etc.), or from recombinant plasmids or viralvectors). The choice of which method to employ will depend upon factorssuch as convenience, desired yield, etc. It is not necessary to employonly one of the above-described methods, processes, or technologies toproduce a particular anti-inflammatory agent; the above-describedprocesses, methods, and technologies may be combined in order to obtaina particular anti-inflammatory agent.

A. Identification of the LFA-1 Binding Partner (ICAM-1)

1. Assays of LFA-1-Dependent Aggregation Many Epstein-Barrvirus-transformed cells exhibit aggregation. This aggregation can beenhanced in the presence of phorbol esters. Such homotypic aggregation(i.e., aggregation involving only one cell type) was found to be blockedby anti-LFA-1 antibodies (Rothlein, R. et al., J. Exper. Med.163:1132-1149 (1986)), which reference is incorporated herein byreference). Thus, the extent of LFA-1-dependent binding may bedetermined by assessing the extent of spontaneous or phorbolester-dependent aggregate formation.

An agent which interferes with LFA-1-dependent aggregation can beidentified through the use of an assay capable of determining whetherthe agent interferes with either the spontaneous, or the phorbolester-dependent aggregation of Epstein-Barr virus-transformed cells.Most Epstein-Barr virus-transformed cells may be employed in such anassay as long as the cells are capable of expressing the LFA-1 receptormolecule. Such cells may be prepared according to the technique ofSpringer, T. A. et al., J. Exper. Med. 160:1901-1918 (1984), whichreference is herein incorporated by reference. Although any such cellmay be employed in the LFA-1 dependent binding assay of the presentinvention, it is preferable to employ cells of the JY cell line(Terhost, C. T. et al., Proc. Natl. Acad. Sci. USA 73:910 (1976)). Thecells may be cultivated in any suitable culture medium; however, it ismost preferable to culture the cells in RMPI 1640 culture mediumsupplemented with 10% fetal calf serum and 50 μg/ml gentamycin (GibcoLaboratories, NY). The cells should be cultured under conditionssuitable for mammalian cell proliferation (i.e., at a temperature ofgenerally 37° C., in an atmosphere of 5% C0₂, at a relative humidity of95%, etc.).

2. LFA-1 Binds to ICAM-1

Human individuals have been identified whose lymphocytes lack the familyof LFA-1 receptor molecules (Anderson, D. C. et al., Fed. Proc.44:2671-2677 (1985); Anderson, D. C. et al., J. Infect. Dis. 152:668-689(1985)). Such individuals are said to suffer from Leukocyte AdhesionDeficiency (LAD). EBV-transformed cells of such individuals fail toaggregate either spontaneously or in the presence of phorbol esters inthe above-described aggregation assay. When such cells are mixed withLFA-1-expressing cells aggregation was observed (Rothlein, R. et al., J.Exper. Med. 163:1132-1149 (1986)) (FIG. 1). Importantly, theseaggregates failed to form if these cells were incubated in the presenceof anti-LFA-1 antibodies. Thus, although the aggregation required LFA-1,the ability of LFA-1-deficient cells to form aggregates withLFA-1-containing cells indicated that the LFA-1 binding partner was notLFA-1 but was rather a previously undiscovered cellular adhesionmolecule. FIG. 1 shows the mechanism of cellular adhesion.

B. Intercellular Adhesion Molecule-1 (ICAM-1)

The novel intercellular adhesion molecule ICAM-1 was first identifiedand partially characterized according to the procedure of Rothlein, R.et al. (J. Immunol. 137:1270-1274 (1986)), which reference is hereinincorporated by reference. To detect the ICAM-1 molecule, monoclonalantibodies were prepared from spleen cells of mice immunized with cellsfrom individuals genetically deficient in LFA-1 expression. Resultantantibodies were screened for their ability to inhibit the aggregation ofLFA-1-expressing cells (FIG. 2). In detail, the ICAM-1 molecule, micewere immunized with EBV-transformed B cells from LAD patients which donot express the LFA-1 antigen. The spleen cells from these animals weresubsequently removed, fused with myeloma cells, and allowed to becomemonoclonal antibody producing hybridoma cells. EBV-transformed B cellsfrom normal individuals which express LFA-1 were then incubated in thepresence of the monoclonal antibody of the hybridoma cell in order toidentify any monoclonal antibody which was capable of inhibiting thephorbol ester mediated, LFA-1 dependent, spontaneous aggregation of theEBV-transformed B cells. Since the hybridoma cells were derived fromcells which had never encountered the LFA-1 antigen no monoclonalantibody to LFA-1 was produced. Hence, any antibody found to inhibitaggregation must be capable of binding to an antigen that, although notLFA-1, participated in the LFA-1 adhesion process. Although any methodof obtaining such monoclonal antibodies may be employed, it ispreferable to obtain ICAM-1-binding monoclonal antibodies by immunizingBALB/C mice using the routes and schedules described by Rothlein, R. etal. (J. Immunol. 137:1270-1274 (1986)) with Epstein-Barrvirus-transformed peripheral blood mononuclear cells from anLFA-1-deficient individuals. Such cells are disclosed by Springer, T.A., et al., (J. Exper. Med. 160:1901-1918 (1984)).

In a preferred method for the generation and detection of antibodiescapable of binding to ICAM-1, mice are immunized with eitherEBV-transformed B cells which express both ICAM-1 and LFA-1 or morepreferably with TNF-activated endothelial cells which express ICAM-1 butnot LFA-1. In a most preferred method for generating hybridoma cellswhich produce anti-ICAM-1 antibodies, a Balb/C mouse was sequentiallyimmunized with JY cells and with differentiated U937 cells (ATCCCRL-1593). The spleen cells from such animals are removed, fused withmyeloma cells and permitted to develop into antibody-producing hybridomacells. The antibodies are screened for their ability to inhibit theLFA-1 dependent, phorbol ester induced aggregation of an EBV transformedcell line, such as JY cells, that expresses both the LFA-1 receptor andICAM-1. As shown by Rothlein, R., et al., (J. Immunol. 137:1270-1274(1987)), antibodies capable of inhibiting such aggregation are thentested for their ability to inhibit the phorbol ester inducedaggregation of a cell line, such as SKW3 (Dustin, M., et al., J. Exper.Med. 165:672-692 (1987)) whose ability to spontaneously aggregate in thepresence of a phorbol ester is inhibited by antibody capable of bindingLFA-1 but is not inhibited by anti-ICAM-1 antibodies. Antibodies capableof inhibiting the phorbol ester induced aggregation of cells such as JYcells, but incapable of inhibiting the phorbol ester induced aggregationof cells such as SKW3 cells are probably anti-ICAM-1 antibodies.Alternatively, antibodies that are capable of binding to ICAM-1 may beidentified by screening for antibodies which are capable of inhibitingthe LFA-1 dependent aggregation of LFA-expression cells (such as JYcells) but are incapable of binding to cells that express LFA-1 butlittle or no ICAM-1 (such as normal granulocytes) or are capable ofbinding to cells that express ICAM-1 but not LFA-1 (such asTNF-activated endothelial cells). Another alternative is toimmunoprecipitate from cells expressing ICAM-1, LFA-1, or both, usingantibodies that inhibit the LFA-1 dependent aggregation of cells, suchas JY cells, and through SDS-PAGE or an equivalent method determine somemolecular characteristic of the molecule precipitated with the antibody.If the characteristic is the same as that of ICAM-1 then the antibodycan be assumed to be an anti-ICAM-1 antibody.

Using monoclonal antibodies prepared in the manner described above, theICAM-1 cell surface molecule was purified, and characterized. ICAM-1 waspurified from human cells or tissue using monoclonal antibody affinitychromatography. In such a method, a monoclonal antibody reactive withICAM-1 is coupled to an inert column matrix. Any method of accomplishingsuch coupling may be employed; however, it is preferable to use themethod of Oettgen, H. C. et al., J. Biol. Chem. 259:12034 (1984)). Whena cellular lysate is passed through the matrix the ICAM-1 moleculespresent are adsorbed and retained by the matrix. By altering the pH orthe ion concentration of the column, the bound ICAM-1 molecules may beeluted from the column. Although any suitable matrix can be employed, itis preferable to employ sepharose (Pharmacia) as the matrix material.The formation of column matrices, and their use in protein purificationare well known in the art.

In a manner understood by those of ordinary skill, the above-describedassays may be used to identify compounds capable of attenuating orinhibiting the rate or extent of cellular adhesion.

ICAM-1 is a cell surface glycoprotein expressed on non-hematopoieticcells such as vascular endothelial cells, thymic epithelial cells,certain other epithelial cells, and fibroblasts, and on hematopoieticcells such as tissue macrophages, mitogen-stimulated T lymphocyteblasts, and germinal centered B cells and dendritic cells in tonsils,lymph nodes, and Peyer's patches. ICAM-1 is highly expressed on vascularendothelial cells in T cell areas in lymph nodes and tonsils showingreactive hyperplasia. ICAM-1 is expressed in low amounts on peripheralblood lymphocytes. Phorbol ester-stimulated differentiation of somemyelomonocytic cell lines greatly increases ICAM-1 expression. Thus,ICAM-1 is preferentially expressed at sites of inflammation, and is notgenerally expressed by quiescent cells. ICAM-1 expression on dermalfibroblasts is increased threefold to fivefold by either interleukin 1or gamma interferon at levels of 10 U/ml over a period of 4 or 10 hours,respectively. The induction is dependent on protein and mRNA synthesisand is reversible.

ICAM-1 displays molecular weight heterogeneity in different cell typeswith a molecular weight of 97 kd on fibroblasts, 114 kd on themyelomonocytic cell line U937, and 90 kd on the B lymphoblastoid cellJY. ICAM-1 biosynthesis has been found to involve an approximately 73 kdintracellular precursor. The non-N-glycosylated form resulting fromtunicamycin treatment (which inhibits glycosylation) has a molecularweight of 55 kd.

ICAM-1 isolated from phorbol ester stimulated U937 cells or fromfibroblast cells yields an identical major product having a molecularweight of 60 kd after chemical deglycosylation. ICAM-1 monoclonalantibodies interfere with the adhesion of phytohaemagglutinin blasts toLFA-1 deficient cell lines. Pretreatment of fibroblasts, but notlymphocytes, with monoclonal antibodies capable of binding ICAM-1inhibits lymphocyte-fibroblast adhesion. Pretreatment of lymphocytes,but not fibroblasts, with antibodies against LFA-1 has also been foundto inhibit lymphocyte-fibroblast adhesion.

ICAM-1 is, thus, the binding ligand of the CD 18 complex on leukocytes.It is inducible on fibroblasts and endothelial cells in vitro byinflammatory mediators such as IL-1, gamma interferon and tumor necrosisfactor in a time frame consistent with the infiltration of lymphocytesinto inflammatory lesions in vivo (Dustin, M. L., et. al., J. Immunol.137:245-254, (1986); Prober, J. S., et. al., J. Immunol. 137:1893-1896,(1986)). Further ICAM-1 is expressed on non-hematopoietic cells such asvascular endothelial cells, thymic epithelial cells, other epithelialcells, and fibroblasts and on hematopoietic cells such as tissuemacrophages, mitogen-stimulated T lymphocyte blasts, and germinal centerB-cells and dendritic cells in tonsils, lymph nodes and Peyer's patches(Dustin, M. L., et. al., J. Immunol. 137:245-254, (1986)). ICAM-1 isexpressed on keratinocytes in benign inflammatory lesions such asallergic eczema, lichen planus, exanthema, urticaria and bullousdiseases. Allergic skin reactions provoked by the application of ahapten on the skin to which the patient is allergic also revealed aheavy ICAM-1 expression on the keratinocytes. On the other hand toxicpatches on the skin did not reveal ICAM-1 expression on thekeratinocytes. ICAM-1 is present on keratinocytes from biopsies of skinlesions from various dermatological disorders and ICAM-1 expression isinduced on lesions from allergic patch tests while keratinocytes fromtoxic patch test lesions failed to express ICAM-1.

ICAM-1 is, therefore, a cellular substrate to which lymphocytes canattach, so that the lymphocytes may migrate to sites of inflammationand/or carry out various effector functions contributing to thisinflammation. Such functions include the production of antibody, lysisof virally infected target cells, etc. The term “inflammation,” as usedherein, is meant to include reactions of the specific and non-specificdefense systems. As used herein, the term “specific defense system” isintended to refer to that component of the immune system that reacts tothe presence of specific antigens. Inflammation is said to result from aresponse of the specific defense system if the inflammation is causedby, mediated by, or associated with a reaction of the specific defensesystem. Examples of inflammation resulting from a response of thespecific defense system include the response to antigens such as rubellavirus, autoimmune diseases, delayed type hypersensitivity responsemediated by T-cells (as seen, for example in individuals who test“positive” in the Mantaux test), etc.

A “non-specific defense system reaction” is a response mediated byleukocytes incapable of immunological memory. Such cells includegranulocytes and macrophages. As used herein, inflammation is said toresult from a response of the non-specific defense system, if theinflammation is caused by, mediated by, or associated with a reaction ofthe non-specific defense system. Examples of inflammation which result,at least in part, from a reaction of the non-specific defense systeminclude inflammation associated with conditions such as: asthma; adultrespiratory distress syndrome (ARDS) or multiple organ injury syndromessecondary to septicemia or trauma; reperfusion injury of myocardial orother tissues; acute glomerulonephritis; reactive arthritis; dermatoseswith acute inflammatory components; acute purulent meningitis or othercentral nervous system inflammatory disorders; thermal injury;hemodialysis; leukopheresis; ulcerative colitis; Crohn's disease;necrotizing enterocolitis; granulocyte trans-fusion associatedsyndromes; and cytokine-induced toxicity.

In accordance with the present invention, ICAM-1 functional derivatives,and especially such derivatives which comprise fragments or mutantvariants of ICAM-1 which possess domains 1, 2 and 3 can be used in thetreatment or therapy of such reactions of the non-specific defensesystem. More preferred for such treatment or therapy are ICAM-1fragments or mutant variants which contain domain 2 of ICAM-1. Mostpreferred for such treatment or therapy are ICAM-1 fragments or mutantvariants which contain domain 1 of ICAM-1.

Functional derivatives of ICAM-1, or a member of the CD18 family, havingup to about 100 residues may be conveniently prepared by in vitrosynthesis. If desired, such fragments may be modified by reactingtargeted amino acid residues of the purified or crude protein with anorganic derivatizing agent that is capable of reacting with selectedside chains or terminal residues. The resulting covalent derivatives maybe used to identify residues important for biological activity. In theembodiments listed below, this aspect of the invention is described withreference to the functional derivatives of ICAM-1. Such methods may alsobe employed to produce functional derivatives of any member of the CD18family of molecules.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidazoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;0-methyl-issurea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using ¹²⁵I or ¹³¹I toprepare labeled proteins for use in radioimmunoassay, the chloramine Tmethod being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′-N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking anICAM-1 functional derivative molecule to a water-insoluble supportmatrix or surface for use in the method for cleaving an ICAM-1functional derivatives fusion polypeptide to release and recover thecleaved polypeptide. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or theonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups.

Functional derivatives of ICAM-1 having altered amino acid sequences canalso be prepared by mutations in the DNA. The nucleotide sequence whichencodes the ICAM-1 gene is shown in FIG. 8. Such variants include, forexample, deletions from, or insertions or substitutions of, residueswithin the amino acid sequence shown in FIG. 8. Any combination ofdeletion, insertion, and substitution may also be made to arrive at thefinal construct, provided that the final construct possesses the desiredactivity. Obviously, the mutations that will be made in the DNA encodingthe variant must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary mRNA structure (see EP Patent Application Publication No.75,444).

At the genetic level, these functional derivatives ordinarily areprepared by site-directed mutagenesis of nucleotides in the DNA encodingthe ICAM-1 molecule, thereby producing DNA encoding the functionalderivative, and thereafter expressing the DNA in recombinant cellculture. The functional derivatives typically exhibit the samequalitative biological activity as the naturally occurring analog. Theymay, however, differ substantially in such characteristics with respectto the normally produced ICAM-1 molecule.

While the site for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed ICAM-1 functional derivatives screened for the optimalcombination of desired activity. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example, site-specific mutagenesis.

Preparation of an ICAM-1 functional derivative molecule in accordanceherewith is preferably achieved by site-specific mutagenesis of DNA thatencodes an earlier prepared functional derivatives or a nonvariantversion of the protein. Site-specific mutagenesis allows the productionof ICAM-1 functional derivatives through the use of specificoligonucleotide sequences that encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a primer sequence of sufficient size and sequence complexity toform a stable duplex on both sides of the deletion junction beingtraversed. Typically, a primer of about 20 to 25 nucleotides in lengthis preferred, with about 5 to 10 residues on both sides of the junctionof the sequence being altered. In general, the technique ofsite-specific mutagenesis is well known in the art, as exemplified bypublications such as Adelman et al., DNA 2:183 (1983), the disclosure ofwhich is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily commercially available and their use is generally well known tothose skilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol. 153:3 (1987)) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant protein. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. (USA) 75:5765 (1978). This primer is thenannealed with the single-stranded protein-sequence-containing vector,and subjected to DNA-polymerizing enzymes such as E. coli polymerase IKlenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform appropriatecells, such as JM101 cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

After such a clone is selected, the mutated protein region may beremoved and placed in an appropriate vector for protein production,generally an expression vector of the type that may be employed fortransformation of an appropriate host.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably 1 to 10 residues, and typically arecontiguous.

Amino acid sequence insertions include amino and/or carboxyl-terminalfusions of from one residue to polypeptides of essentially unrestrictedlength, as well as intrasequence insertions of single or multiple aminoacid residues. Intrasequence insertions (i.e., insertions within thecomplete ICAM-1 molecule sequence) may range generally from about 1 to10 residues, more preferably 1 to 5. An example of a terminal insertionincludes a fusion of a signal sequence, whether heterologous orhomologous to the host cell, to the N-terminus of the molecule tofacilitate the secretion of the ICAM-1 functional derivative fromrecombinant hosts.

The third group of functional derivatives are those in which at leastone amino acid residue in the ICAM-1 molecule, and preferably, only one,has been removed and a different residue inserted in its place. Suchsubstitutions preferably are made in accordance with the following Tablewhen it is desired to modulate finely the characteristics of the ICAM-1molecule.

TABLE 1 Original Residue Exemplary Substitutions Ala gly; ser Arg lysAsn gln; his Asp glu Cys ser Gln asn Glu asp Gly ala; pro His asn; glnIle leu; val Leu ile; val Lys arg; gln; glu Met leu; tyr; ile Phe met;leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Substantial changes in functional or immunological identity are made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions thatin general are expected to those in which (a) glycine and/or proline issubstituted by another amino acid or is deleted or inserted; (b) ahydrophilic residue, e.g., seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, oralanyl; (c) a cysteine residue is substituted for (or by) any otherresidue; (d) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) a residue havingan electronegative charge, e.g., glutamyl or aspartyl; or (e) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having such a side chain, e.g., glycine.

Most deletions and insertions, and substitutions in particular, are notexpected to produce radical changes in the characteristics of the ICAM-1molecule. However, when it is difficult to predict the exact effect ofthe substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays. For example, a functional derivative typicallyis made by site-specific mutagenesis of the native ICAM-1molecule-encoding nucleic acid, expression of the variant nucleic acidin recombinant cell culture, and, optionally, purification from the cellculture, for example, by immunoaffinity adsorption on an anti-ICAM-1molecule antibody column (to absorb the functional derivative by bindingit to at least one remaining immune epitope).

The activity of the cell lysate or purified ICAM-1 molecule functionalderivative is then screened in a suitable screening assay for thedesired characteristic. For example, a change in the immunologicalcharacter of the functional derivative, such as affinity for a givenantibody, is measured by a competitive type immunoassay. Changes inimmunomodulation activity are measured by the appropriate assay.Modifications of such protein properties as redox or thermal stability,biological half-life, hydrophobicity, susceptibility to proteolyticdegradation or the tendency to aggregate with carriers or into multimersare assayed by methods well known to the ordinarily skilled artisan.

C. Cloning of the ICAM-1 Gene

Any of a variety of procedures may be used to clone the ICAM-1 gene. Onesuch method entails analyzing a shuttle vector library of cDNA inserts(derived from an ICAM-1 expressing cell) for the presence of an insertwhich contains the ICAM-1 gene. Such an analysis may be conducted bytransfecting cells with the vector and then assaying for ICAM-1expression. The preferred method for cloning this gene entailsdetermining the amino acid sequence of the ICAM-1 molecule. Toaccomplish this task ICAM-1 protein may be purified and analyzed byautomated sequenators. Alternatively, the molecule may be fragmented aswith cyanogen bromide, or with proteases such as papain, chymotrypsin ortrypsin (Oike, Y. et al., J. Biol. Chem. 257:9751-9758 (1982); Liu, C.et al., Int. J. Pept. Protein Res. 21:209-215 (1983)). Although it ispossible to determine the entire amino acid sequence of ICAM-1, it ispreferable to determine the sequence of peptide fragments of themolecule. If the peptides are greater than 10 amino acids long, thesequence information is generally sufficient to permit one to clone agene such as the gene for ICAM-1.

The sequence of amino acid residues in a peptide is designated hereineither through the use of their commonly employed 3-letter designationsor by their single-letter designations. A listing of these 3-letter and1-letter designations may be found in textbooks such as Biochemistry,Lehninger, A., Worth Publishers, New York, N.Y. (1970). When such asequence is listed vertically, the amino terminal residue is intended tobe at the top of the list, and the carboxy terminal residue of thepeptide is intended to be at the bottom of the list. Similarly, whenlisted horizontally, the amino terminus is intended to be on the leftend whereas the carboxy terminus is intended to be at the right end. Theresidues of amino acids in a peptide may be separated by hyphens. Suchhyphens are intended solely to facilitate the presentation of asequence. As a purely illustrative example, the amino acid sequencedesignated:

Gly-Ala-Ser-Phe

indicates that an Ala residue is linked to the carboxy group of Gly, andthat a Ser residue is linked to the carboxy group of the Ala residue andto the amino group of a Phe residue. The designation further indicatesthat the amino acid sequence contains the tetrapeptide Gly-Ala-Ser-Phe.The designation is not intended to limit the amino acid sequence to thisone tetrapeptide, but is intended to include (1) the tetrapeptide havingone or more amino acid residues linked to either its amino or carboxyend, (2) the tetrapeptide having one or more amino acid residues linkedto both its amino and its carboxy ends, (3) the tetrapeptide having noadditional amino acid residues.

Once one or more suitable peptide fragments have been sequenced, the DNAsequences capable of encoding them are examined. Because the geneticcode is degenerate, more than one codon may be used to encode aparticular amino acid (Watson, J. D., In: Molecular Biology of the Gene,3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977), pp. 356-357).The peptide fragments are analyzed to identify sequences of amino acidswhich may be encoded by oligonucleotides having the lowest degree ofdegeneracy. This is preferably accomplished by identifying sequencesthat contain amino acids which are encoded by only a single codon.Although occasionally such amino acid sequences may be encoded by only asingle oligonucleotide, frequently the amino acid sequence can beencoded by any of a set of similar oligonucleotides. Importantly,whereas all of the members of the set contain oligonucleotides which arecapable of encoding the peptide fragment and, thus, potentially containthe same nucleotide sequence as the gene which encodes the peptidefragment, only one member of the set contains a nucleotide sequence thatis identical to the nucleotide sequence of this gene. Because thismember is present within the set, and is capable of hybridizing to DNAeven in the presence of the other members of the set, it is possible toemploy the unfractionated set of oligonucleotides in the same manner inwhich one would employ a single oligonucleotide to clone the gene thatencodes the peptide.

In a manner exactly analogous to that described above, one may employ anoligonucleotide (or set of oligonucleotides) which have a nucleotidesequence that is complementary to the oligonucleotide sequence or set ofsequences that is capable of encoding the peptide fragment.

A suitable oligonucleotide, or set of oligonucleotides which is capableof encoding a fragment of the ICAM-1 gene (or which is complementary tosuch an oligonucleotide, or set of oligonucleotides) is identified(using the above-described procedure), synthesized, and hybridized, bymeans well known in the art, against a DNA or, more preferably, a cDNApreparation derived from human cells which are capable of expressingICAM-1 gene sequences. Techniques of nucleic acid hybridization aredisclosed by Maniatis, T. et al., In: Molecular Cloning, a LaboratoryManual, Coldspring Harbor, N.Y. (1982), and by Haymes, B. D. et al., In:Nucleic Acid Hybrization, a Practical Approach, IRL Press, Washington,D.C. (1985), which references are herein incorporated by reference. Thesource of DNA or cDNA used will preferably have been enriched for ICAM-1sequences. Such enrichment can most easily be obtained from cDNAobtained by extracting RNA from cells cultured under conditions whichinduce ICAM-1 synthesis (such as U937 grown in the presence of phorbolesters, etc.).

Techniques such as, or similar to, those described above havesuccessfully enabled the cloning of genes for human aldehydedehydrogenases (Hsu, L. C. et al., Proc. Natl. Acad. Sci. USA82:3771-3775 (1985)), fibronectin (Suzuki, S. et al., Eur. Mol. Biol.Organ. J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter,P. et al., Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-typeplasminogen activator (Pennica, D. et al., Nature 301:214-221 (1983))and human term placental alkaline phosphatase complementary DNA (Kam, W.et al., Proc. Natl. Acad. Sci. USA 82:8715-8719 (1985)).

In a preferred alternative way of cloning the ICAM-1 gene, a library ofexpression vectors is prepared by cloning DNA or, more preferably cDNA,from a cell capable of expressing ICAM-1 into an expression vector. Thelibrary is then screened for members capable of expressing a proteinwhich binds to anti-ICAM-1 antibody, and which has a nucleotide sequencethat is capable of encoding polypeptides that have the same amino acidsequence as ICAM-1 or fragments of ICAM-1.

The cloned ICAM-1 gene, obtained through the methods described above,may be operably linked to an expression vector, and introduced intobacterial, or eukaryotic cells to produce ICAM-1 protein. Techniques forsuch manipulations are disclosed by Maniatis, T. et al., supra, and arewell known in the art.

D. Uses of Assays of LFA-1 Dependent Aggregation

The above-described assay, capable of measuring LFA-1 dependentaggregation, may be employed to identify agents which act as antagoniststo inhibit the extent of LFA-1 dependent aggregation. Such antagonistsmay act by impairing the ability of LFA-1 or of ICAM-1 to mediateaggregation. Thus, such agents include immunoglobulins such as anantibody capable of binding to either LFA-1 or ICAM-1. Additionally,non-immunoglobulin (i.e., chemical) agents may be examined, using theabove-described assay, to determine whether they are antagonists ofLFA-1 aggregation.

E. Uses of Antibodies Capable of Binding to ICAM-1 Receptor Proteins

1. Anti-Inflammatory Agents

Monoclonal antibodies to members of the CD 18 complex inhibit manyadhesion dependent functions of leukocytes including binding toendothelium (Haskard, D., et al., J. Immunol. 137:2901-2906 (1986)),homotypic adhesions (Rothlein, R., et al., J. Exp. Med. 163:1132-1149(1986)), antigen and mitogen induced proliferation of lymphocytes(Davignon, D., et al., Proc. Natl. Acad. Sci. USA 78:4535-4539 (1981)),antibody formation (Fischer, A., et al., J. Immunol. 136:3198-3203(1986)), and effector functions of all leukocytes such as lytic activityof cytotoxic T cells (Krensky, A. M., et al., J. Immunol. 132:2180-2182(1984)), macrophages (Strassman, G., et al., J. Immunol. 136:4328-4333(1986)), and all cells involved in antibody-dependent cellularcytotoxicity reactions (Kohl, S., et al., J. Immunol. 133:2972-2978(1984)). In all of the above functions, the antibodies inhibit theability of the leukocyte to adhere to the appropriate cellular substratewhich in turn inhibits the final outcome. Although both polyclonal andmonoclonal antibodies may be employed in accordance with the invention,monoclonal antibodies are especially preferred for such use.

As discussed above, the binding of ICAM-1 molecules to the members ofLFA-1 family of molecules is of central importance in cellular adhesion.Through the process of adhesion, lymphocytes are capable of continuallymonitoring an animal for the presence of foreign antigens. Althoughthese processes are normally desirable, they are also the cause of organtransplant rejection, tissue graft rejection and many autoimmunediseases. Hence, any means capable of attenuating or inhibiting cellularadhesion would be highly desirable in recipients of organ transplants,tissue grafts or autoimmune patients.

Monoclonal and polyclonal antibodies capable of binding to ICAM-1 arehighly suitable as anti-inflammatory agents in a mammalian subject.Monoclonal antibodies are especially preferred for such use.Significantly, such agents differ from general anti-inflammatory agentsin that they are capable of selectively inhibiting adhesion, and do notoffer other side effects such as nephrotoxicity which are found withconventional agents. Monoclonal antibodies capable of binding to ICAM-1can therefore be used to prevent organ or tissue rejection, or modifyautoimmune responses without the fear of such side effects, in themammalian subject.

Importantly, the use of monoclonal antibodies capable of recognizingICAM-1 may permit one to perform organ transplants even betweenindividuals having HLA mismatch.

As indicated above, both polyclonal and monoclonal antibodies may beemployed in accordance with the present invention. Of special interestto the present invention are antibodies to ICAM-1 (or their functionalderivatives), or to members of the CD18 family (or their functionalderivatives), which are produced in humans, or are “humanized” (i.e.non-immunogenic in a human) by recombinant or other technology. Suchantibodies are the equivalents of the monoclonal and polyclonalantibodies disclosed herein, but are less immunogenic, and are bettertolerated by the patient.

Humanized antibodies may be produced, for example by replacing animmunogenic portion of an antibody with a corresponding, butnon-immunogenic portion (i.e. chimeric antibodies) (Robinson, R. R. etal., International Patent Publication PCT/US86/02269; Akira, K. et al.,European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison, S. L. et al., European Patent Application173,494; Neuberger, M. S. et al., PCT Application WO 86/01533; Cabilly,S. et al., European Patent Application 125,023; Better, M. et al.,Science 240:1041-1043 (1988); Liu, A. Y. et al., Proc. Natl. Acad. Sci.USA 84:3439-3443 (1987); Liu, A. Y. et al., J. Immunol. 139:3521-3526(1987); Sun, L. K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987);Nishimura, Y. et al., Canc. Res. 47:999-1005 (1987); Wood, C. R. et al.,Nature 314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst.80:1553-1559 (1988); all of which references are incorporated herein byreference). General reviews of “humanized” chimeric antibodies areprovided by Morrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi,V. T. et al., BioTechnioues 4:214 (1986); which references areincorporated herein by reference).

Suitable “humanized” antibodies can be alternatively produced by CDR orCEA substitution (Jones, P. T. et al., Nature 321:552-525 (1986);Verhoeyan et al., Science 239:1534 (1988); Beidler, C. B. et al., J.Immunol. 141:4053-4060 (1988); all of which references are incorporatedherein by reference).

2. Suppressors of Delayed Type Hypersensitivity Reaction

Since ICAM-1 molecules are expressed mostly at sites of inflammation,such as those sites involved in delayed type hypersensitivity reaction,antibodies (especially monoclonal antibodies) capable of binding toICAM-1 molecules have therapeutic potential in the attenuation orelimination of such reactions. This potential therapeutic use may beexploited in either of two manners. First, a composition containing amonoclonal antibody against ICAM-1 may be administered to a patientexperiencing delayed type hypersensitivity reaction. For example, suchcompositions might be provided to a individual who had been in contactwith antigens such as poison ivy, poison oak, etc. In the secondembodiment, the monoclonal antibody capable of binding to ICAM-1 isadministered to a patient in conjunction with an antigen in order toprevent a subsequent inflammatory reaction. Thus, the additionaladministration of an antigen with an ICAM-1-binding monoclonal antibodymay temporarily tolerize an individual to subsequent presentation ofthat antigen.

3. Therapy for Chronic Inflammatory Disease

Since LAD patients that lack LFA-1 do not mount an inflammatoryresponse, it is believed that antagonism of LFA-1's natural ligand,ICAM-1, will also inhibit an inflammatory response. The ability ofantibodies against ICAM-1 to inhibit inflammation provides the basis fortheir therapeutic use in the treatment of chronic inflammatory diseasesand autoimmune diseases such as lupus erythematosus, autoimmunethyroiditis, experimental allergic encephalomyelitis (EAE), multiplesclerosis, some forms of diabetes Reynaud's syndrome, rheumatoidarthritis, etc. Such antibodies may also be employed as a therapy in thetreatment of psoriasis. In general, the monoclonal antibodies capable ofbinding to ICAM-1 may be employed in the treatment of those diseasescurrently treatable through steroid therapy.

4. Diagnostic and Prognostic Applications

Since ICAM-1 is expressed mostly at sites of inflammation, monoclonalantibodies capable of binding to ICAM-1 may be employed as a means ofimaging or visualizing the sites of infection and inflammation in apatient. In such a use, the monoclonal antibodies are detectablylabeled, through the use of radioisotopes, affinity labels (such asbiotin, avidin, etc.) fluorescent labels, paramagnetic atoms, etc.Procedures for accomplishing such labeling are well known to the art.Clinical application of antibodies in diagnostic imaging are reviewed byGrossman, H. B., Urol. Clin. North Amer. 13:465-474 (1986)), Unger, E.C. et al., Invest. Radiol. 20:693-700 (1985)), and Khaw, B. A. et al.,Science 209:295-297 (1980)).

The presence of inflammation may also be detected through the use ofbinding ligands, such as mRNA, cDNA, or DNA which bind to ICAM-1 genesequences, or to ICAM-1 mRNA sequences, of cells which express ICAM-1.Techniques for performing such hybridization assays are described byManiatais, T. (supra).

The detection of foci of such detectably labeled antibodies isindicative of a site of inflammation or tumor development. In oneembodiment, this examination for inflammation is done by removingsamples of tissue or blood and incubating such samples in the presenceof the detectably labeled antibodies. In a preferred embodiment, thistechnique is done in a non-invasive manner through the use of magneticimaging, fluorography, etc. Such a diagnostic test may be employed inmonitoring organ transplant recipients for early signs of potentialtissue rejection. Such assays may also be conducted in efforts todetermine an individual's predilection to rheumatoid arthritis or otherchronic inflammatory diseases.

5. Adjunct to the Introduction of Antigenic Material Administered forTherapeutic or Diagnostic Purposes

Immune responses to therapeutic or diagnostic agents such as, forexample, bovine insulin, interferon, tissue-type plasminogen activatoror murine monoclonal antibodies substantially impair the therapeutic ordiagnostic value of such agents, and can, in fact, causes diseases suchas serum sickness. Such a situation can be remedied through the use ofthe antibodies of the present invention. In this embodiment, suchantibodies would be administered in combination with the therapeutic ordiagnostic agent. The addition of the antibodies prevents the recipientfrom recognizing the agent, and therefore prevents the recipient frominitiating an immune response against it. The absence of such an immuneresponse results in the ability of the patient to receive additionaladministrations of the therapeutic or diagnostic agent.

F. Uses of Intercellular Adhesion Molecule-1 (ICAM-1)

ICAM-1 is a binding partner of LFA-1. As such, ICAM-1 or its functionalderivatives may be employed interchangeably with antibodies capable ofbinding to LFA-1 in the treatment of disease. Thus, in solubilized form,such molecules may be employed to inhibit inflammation, organ rejection,graft rejection, etc. ICAM-1, or its functional derivatives may be usedin the same manner as anti-ICAM antibodies to decrease theimmunogenicity of therapeutic or diagnostic agents.

ICAM-1, its functional derivatives, and its antagonists may be used toblock the metastasis or proliferation of tumor cells which expresseither ICAM-1 or LFA-1 on their surfaces. A variety of methods may beused to accomplish such a goal. For example, the migration ofhematopoietic cells requires LFA-1-ICAM-1 binding. Antagonists of suchbinding therefore suppress this migration and block the metastasis oftumor cells of leukocyte lineage. Alternatively, toxin-derivatizedmolecules, capable of binding either ICAM-1 or a member of the LFA-1family of molecules, may be administered to a patient. When suchtoxin-derivatized molecules bind to tumor cells that express ICAM-1 or amember of the LFA-1 family of molecules, the presence of the toxin killsthe tumor cell thereby inhibiting the proliferation of the tumor.

G. Uses of Non-Immunoglobulin Antagonists of ICAM-1 Dependent Adhesion

ICAM-1-dependent adhesion can be inhibited by non-immunoglobulinantagonists which are capable of binding to either ICAM-1 or to LFA-1.One example of a non-immunoglobulin antagonist of ICAM-1 is LFA-1. Anexample of a non-immunoglobulin antagonist which binds to LFA-1 isICAM-1. Through the use of the above-described assays, additionalnon-immunoglobulin antagonists can be identified and purified.

One especially preferred class of non-immunological antagonists comprisesoluble derivatives of ICAM-1, CD11a, CD11b, CD11c, CD18 molecules, orof the CD11a/CD18, CD11b/CD18, or CD11c/CD18 heterodimers. The solublederivatives referred to above are derivatives which are not bound to amembrane of a cell. Such derivatives may comprise truncated moleculeswhich lack a transmembrane domain. Alternatively, they may comprisemutant forms of the natural molecules which lack the capacity to bebound (or stably bound) to the membrane of a cell even though theycontain a transmembrane domain. Soluble derivatives of ICAM-1 and theirpreparation are disclosed by Marlin, S. D. et al., Nature 344:70-72(1990), which reference is incorporated herein by reference). Among thepreferred functional derivatives of the present invention are solublefragments of the ICAM-1 molecule which contain domains 1, 2, and 3 ofICAM-1. More preferred are soluble fragments of the ICAM-1 moleculewhich contain domains 1 and 2 of ICAM-1. Most preferred are solublefragments of the ICAM-1 molecule which contain domain 1 of ICAM-1.

Non-immunoglobulin antagonists of ICAM-1 dependent adhesion may be usedfor the same purpose as antibodies to LFA-1 or antibodies to ICAM-1.

H. Administration of the Compositions of the Present Invention

The therapeutic effects of ICAM-1 may be obtained by providing to apatient the entire ICAM-1 molecule, or any therapeutically activepeptide fragments thereof.

ICAM-1 and its functional derivatives may be obtained eithersynthetically, through the use of recombinant DNA technology, or byproteolysis. The therapeutic advantages of ICAM-1 may be augmentedthrough the use of functional derivatives of ICAM-1 possessingadditional amino acid residues added to enhance coupling to carrier orto enhance the activity of the ICAM-1. The scope of the presentinvention is further intended to include functional derivatives ofICAM-1 which lack certain amino acid residues, or which contain alteredamino acid residues, so long as such derivatives exhibit the capacity toaffect cellular adhesion.

Both the antibodies of the present invention and the ICAM-1 moleculedisclosed herein are said to be “substantially free of naturalcontaminants” if preparations which contain them are substantially freeof materials with which these products are normally and naturally found.

The present invention extends to antibodies, and biologically activefragments thereof, (whether polyclonal or monoclonal) which are capableof binding to ICAM-1. Such antibodies may be produced either by ananimal, or by tissue culture, or recombinant DNA means.

In providing a patient with antibodies, or fragments thereof, capable ofbinding to ICAM-1, or when providing ICAM-1 (or a fragment, variant, orderivative thereof) to a recipient patient, the dosage of administeredagent will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition, previous medicalhistory, etc. In general, it is desirable to provide the recipient witha dosage of antibody which is in the range of from about 1 pg/kg to 10mg/kg (body weight of patient), although a lower or higher dosage may beadministered. When providing ICAM-1 molecules or their functionalderivatives to a patient, it is preferable to administer such moleculesin a dosage which also ranges from about 1 pg/kg to 10 mg/kg (bodyweight of patient) although a lower or higher dosage may also beadministered. As discussed below, the therapeutically effective dose canbe lowered if the anti-ICAM-1 antibody is additionally administered withan anti-LFA-1 antibody. As used herein, one compound is said to beadditionally administered with a second compound when the administrationof the two compounds is in such proximity of time that both compoundscan be detected at the same time in the patient's serum.

Both the antibody capable of binding to ICAM-1 and ICAM-1 itself may beadministered to patients intravenously, intramuscularly, subcutaneously,enterally, or parenterally. When administering antibody or ICAM-1 byinjection, the administration may be by continuous infusion, or bysingle or multiple boluses.

The anti-inflammatory agents of the present invention are intended to beprovided to recipient subjects in an amount sufficient to suppressinflammation. An amount is said to be sufficient to “suppress”inflammation if the dosage, route of administration, etc. of the agentare sufficient to attenuate or prevent inflammation.

Anti-ICAM-1 antibody, or a fragment thereof, may be administered eitheralone or in combination with one or more additional immunosuppressiveagents (especially to a recipient of an organ or tissue transplant). Theadministration of such compound(s) may be for either a “prophylactic” or“therapeutic” purpose. When provided prophylactically, theimmunosuppressive compound(s) are provided in advance of anyinflammatory response or symptom (for example, prior to, at, or shortlyafter) the time of an organ or tissue transplant but in advance of anysymptoms of organ rejection). The prophylactic administration of thecompound(s) serves to prevent or attenuate any subsequent inflammatoryresponse (such as, for example, rejection of a transplanted organ ortissue, etc.). When provided therapeutically, the immunosuppressivecompound(s) is provided at (or shortly after) the onset of a symptom ofactual inflammation (such as, for example, organ or tissue rejection).The therapeutic administration of the compound(s) serves to attenuateany actual inflammation (such as, for example, the rejection of atransplanted organ or tissue).

The anti-inflammatory agents of the present invention may, thus, beprovided either prior to the onset of inflammation (so as to suppress ananticipated inflammation) or after the initiation of inflammation.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

The antibody and ICAM-1 molecules of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby these materials, or their functional derivatives,are combined in admixture with a pharmaceutically acceptable carriervehicle. Suitable vehicles and their formulation, inclusive of otherhuman proteins, e.g., human serum albumin, are described, for example,in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack,Easton Pa. (1980)). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of anti-ICAM antibody or ICAM-1molecule, or their functional derivatives, together with a suitableamount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb anti-ICAM-1 antibody or ICAM-1,or their functional derivatives. The controlled delivery may beexercised by selecting appropriate macromolecules (for examplepolyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine, sulfate) and the concentration of macromolecules as well asthe methods of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate anti-ICAM-1 antibody or ICAM-1 molecules,or their functional derivatives, into particles of a polymeric materialsuch as polyesters, polyamino acids, hydrogels, poly(lactic acid) orethylene vinylacetate copolymers. Alternatively, instead ofincorporating these agents into polymeric particles, it is possible toentrap these materials in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatine-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

Example 1

Culturing of Mammalian Cells

In general, the EBV-transformed and hybridoma cells of the presentinvention were maintained in RMPI 1640 culture medium, supplemented with20 mM L-glutamine, 50 pg/ml gentamicin, and 10% fetal bovine (or fetalcalf) sera. Cells were cultured at 37° C. in a 5% CO₂, 95% air humidityatmosphere.

To establish Epstein-Barr virus (EBV) transformants, 106 T cell depletedperipheral blood mononuclear cells/ml in RPMI 1640 medium supplementedwith 20% fetal calf serum (FCS), and 50 pg/ml gentamicin were incubatedfor 16 hours with EBV-containing supernatant of B95-8 cells(Thorley-Lawson, D. A. et al., J. Exper. Med. 146:495 (1977)). Cells in0.2 ml aliquot were placed in 10 microtiter wells. Medium was replacedwith RPMI 1640 medium (supplemented with 20% fetal calf serum and 50μg/ml gentamicin) until cell growth was noted. Cells grew in most wellsand were expanded in the same medium. Phytohemagglutinin (PHA) blastswere established at 10⁶ cells/ml in RPMI 1640 medium (supplemented with20% fetal calf serum) containing a 1:800 dilution of PHA-P (DifcoLaboratories, Inc., Detroit, Mich.). PHA lines were expanded withinterleukin 2 (IL-2)-conditioned medium and pulsed weekly with PHA(Cantrell, D. A. et al., J. Exper. Med. 158:1895 (1983)). The aboveprocedure was disclosed by Springer, T. et al., J. Exper. Med.160:1901-1918 (1984), which reference is herein incorporated byreference. Cells obtained through the above procedure are then screenedwith anti-LFA-1 antibodies to determine whether they express the LFA-1antigen. Such antibodies are disclosed by Sanchez-Madrid, F. et al., J.Exper. Med. 158:1785 (1983).

Example 2

Assays of Cellular Aggregation and Adhesion

In order to assess the extent of cellular adhesion, aggregation assayswere employed. Cell lines used in such assays were washed two times withRPMI 1640 medium containing 5 mM Hepes buffer (Sigma Chemical Co., St.Louis) and resuspended to a concentration of 2×10⁶ cells/ml. Added toflat-bottomed, 96-well microtiter plates (No. 3596; Costar, Cambridge,Mass.) were 50 μl of appropriate monoclonal antibody supernatant or 50μl of complete medium with or without purified monoclonal antibodies, 50μl of complete medium containing 200 ng/ml of the phorbol ester phorbolmyristate acetate (PMA) and 100 μl of cells at a concentration of 2×10⁶cells/ml in complete medium. This yielded a final concentration of 50ng/ml PMA and 2×10⁵ cells/well. Cells were allowed to settlespontaneously, and the degree of aggregation was scored at various timepoints. Scores ranged from 0 to 5+, where 0 indicated that essentiallyno cells were in clusters; 1+ indicated that less than 10% of the cellswere in aggregates; 2+ indicated that less than 507% of the cells wereaggregated; 3+ indicated that up to 100% of the cells were in small,loose clusters; 4+ indicated that up to 100% of the cells wereaggregated in larger clusters; and 5+ indicated that 100% of the cellswere in large, very compact aggregates. In order to obtain a morequantitative estimate of cellular adhesion, reagents and cells wereadded to 5 ml polystyrene tubes in the same order as above. Tubes wereplaced in a rack on a gyratory shaker at 37° C. After 1 hour atapproximately 200 rpm, 10 μl of the cell suspension was placed in ahemocytometer and the number of free cells was quantitated. Percentaggregation was determined by the following equation:

${\% \mspace{14mu} {aggregation}} = {100 \times \left( {1 - \frac{{number}\mspace{14mu} {of}\mspace{14mu} {free}\mspace{14mu} {cells}}{{number}\mspace{14mu} {of}\mspace{14mu} {input}\mspace{14mu} {cells}}} \right)}$

The number of input cells in the above formula is the number of cellsper ml in a control tube containing only cells and complete medium thathad not been incubated. The number of free cells in the above equationequals the number of non-aggregated cells per ml from experimentaltubes. The above procedures were described by Rothlein, R., et al., J.Exper. Med. 163:1132-1149 (1986).

Example 3 LFA-1 Dependent Cellular Aggregation

The qualitative aggregation assay described in Example 2 was performedusing the Epstein-Barr transformed cell line JY. Upon addition of PMA tothe culture medium in the microtiter plates, aggregation of cells wasobserved. Time lapse video recordings showed that the JY cells on thebottom of the microtiter wells were motile and exhibited active membraneruffling and pseudopodia movement. Contact between the pseudopodia ofneighboring cells often resulted in cell-cell adherence. If adherencewas sustained, the region of cell contact moved to the uropod. Contactcould be maintained despite vigorous cell movements and the tugging ofthe cells in opposite directions. The primary difference betweenPMA-treated and untreated cells appeared to be in the stability of thesecontacts once they were formed. With PMA, clusters of cells developed,growing in size as additional cells adhered at their periphery.

As a second means of measuring adhesion, the quantitative assaydescribed in Example 2 was used. Cell suspensions were shaken at 200 rpmfor 2 hours, transferred to a hemocytometer, and cells not in aggregateswere enumerated. In the absence of PMA, 42% (SD=20%, N=6) of JY cellswere in aggregates after 2 hours, while JY cells incubated underidentical conditions with 50 ng/ml of PMA had 87% (SD=8%, N=6) of thecells in aggregates. Kinetic studies of aggregation showed that PMAenhanced the rate and magnitude of aggregation at all time points tested(FIG. 3).

Example 4 Inhibition of Aggregation of Cells sing Anti-LFA-1 MonoclonalAntibodies

To examine the effects of anti-LFA-1 monoclonal antibodies onPMA-induced cellular aggregation, such antibodies were added to cellsincubated in accordance with the qualitative aggregation assay ofExample 2. The monoclonal antibodies were found to inhibit the formationof aggregates of cells either in the presence or absence of PMA. Boththe F(ab′)₂ and Fab′ fragments of monoclonal antibodies against thealpha chain of LFA-1 were capable of inhibiting cellular aggregation.Whereas essentially 100% of cells formed aggregates in the absence ofanti-LFA-1 antibody, less than 20% of the cells were found to be inaggregates when antibody was added. The results of this experiment weredescribed by Rothlein, R. et al. (J. Exper. Med. 163:1132-1149 (1986).

Example 5 Cellular Aggregation Requires the LFA-1 Receptor

EBV-transformed lymphoblastoid cells were prepared from patients in themanner described in Example 1. Such cells were screened againstmonoclonal antibodies capable of recognizing LFA-1 and the cells werefound to be LFA-1 deficient.

The qualitative aggregation assay described in Example 2 was employed,using the LFA-1 deficient cells described above. Such cells failed tospontaneously aggregate, even in the presence of PMA.

Example 6 The Discovery of ICAM-1

The LFA-1 deficient cells of Example 5 were labeled withcarboxyfluorescein diacetate (Patarroyo, M. et al., Cell. Immunol.63:237-248 (1981)). The labeled cells were mixed in a ratio of 1:10 withautologous or JY cells and the percentage of fluorescein-labeled cellsin aggregates was determined according to the procedure of Rothlein, R.et al., J. Exper. Med. 163:1132-1149 (1986). The LFA-1 deficient cellswere found to be capable of congregating with LFA-1 expressing cells(FIG. 4).

To determine whether LFA-1 was important only in forming aggregates, orin their maintenance, antibodies capable of binding to LFA-1 were addedto the preformed aggregates described above. The addition of antibodywas found to strongly disrupt the preformed aggregation. Time lapsevideo recording confirmed that addition of the monoclonal antibodies topreformed aggregates began to cause disruption within 2 hours (Table 2).After addition of monoclonal antibodies against LFA-1, pseudopodialmovements and changes in shape of individual cells within aggregatescontinued unchanged. Individual cells gradually disassociated from theperiphery of the aggregate; by 8 hours cells were mostly dispersed. Byvideo time lapse, the disruption of pre-formed aggregates by LFA-1monoclonal antibodies appeared equivalent to the aggregation process inthe absence of LFA-1 monoclonal antibody running backwards in time.

TABLE 2 Ability of Anti-LFA-1 Monoclonal Antibodies to Disrupt PreformedPMA-Induced JY Cell Aggregates Aggregation score 18 h Exp. 2 h^(a) −mAb+mAb 1 4+ 4+ 1+^(b) 2 3+ 4+ 1+^(c) 3 5+ 5+ 1+^(d) Aggregation in thequalitative microtiter plate assay was scored visually. With anti-LFA-1present throughout the assay period, aggregation was less than 1+.^(a)Amount of aggregation just before addition of Mono-clonal antibodyat 2 h. ^(b)TS1/18 + TS1/22. ^(c)TS1/18. ^(d)TS1/22.

Example 7 The Requirement of Divalent Ions for LFA-1 DependentAggregation

LFA-1 dependent adhesions between cytotoxic T cells and targets requirethe presence of magnesium (Martz, E. J. Cell. Biol. 84:584-598 (1980)).PMA-induced JY cell aggregation was tested for divalent cationdependence. JY cells failed to aggregate (using the assay of Example 2)in medium free of calcium or magnesium ions. The addition of divalentmagnesium supported aggregation at concentrations as low as 0.3 mM.Addition of calcium ions alone had little effect. Calcium ions, however,were found to augment the ability of magnesium ions to supportPMA-induced aggregation. When 1.25 mM calcium ions were added to themedium, magnesium ion concentrations as low as 0.02 millimolar werefound to support aggregation. These data show that the LFA-1 dependentaggregation of cells requires magnesium ions, and that calcium ions,though insufficient of themselves, can synergize with magnesium ions topermit aggregation.

Example 8 The Isolation of Hybridoma Cells Capable of ExpressingAnti-ICAM-1 Monoclonal Antibodies

Monoclonal antibodies capable of binding to ICAM-1 were isolatedaccording to the method of Rothlein, R. et al., J. Immunol.137:1270-1274 (1986), which reference has been incorporated by referenceherein. Thus, 3 BALB/C mice were immunized intraperitoneally withEBV-transformed peripheral blood mononuclear cells from anLFA-1-deficient individual (Springer, T. A. et al., J. Exper. Med.160:1901 (1984)). Approximately 10⁷ cells in 1 ml RPMI 1640 medium wasused for each immunization. The immunizations were administered 45, 29,and 4 days before spleen cells were removed from the mice in order toproduce the desired hybridoma cell lines. On day 3 before the removal ofthe spleen cells, the mice were given an additional 10⁷ cells in 0.15 mlmedium (intravenously).

Isolated spleen cells from the above-described animals were fused withP3X73Ag8.653 myeloma cells at a ratio of 4:1 according to the protocolof Galfre, G. et al., Nature 266:550 (1977). Aliquots of the resultinghybridoma cells were introduced into 96-well microtiter plates. Thehybridoma supernatants were screened for inhibition of aggregation, andone inhibitory hybridoma (of over 600 wells tested) was cloned andsubcloned by limiting dilution. This subclone was designated RR1/1.1.1(hereinafter designated “RR 1/1”).

Monoclonal antibody RR1/1 was consistently found to inhibitPMA-stimulated aggregation of the LFA-1 expressing cell line JY. TheRR1/1 monoclonal antibody inhibited aggregation equivalently, orslightly less than some monoclonal antibodies to the LFA-1 alpha or betasubunits. In contrast, control monoclonal antibody against HLA, which isabundantly expressed on JY cells, did not inhibit aggregation. Theantigen bound by monoclonal antibody RR1/1 is defined as theintercellular adhesion molecule-1 (ICAM-1).

Example 9 Use of Anti-ICAM-1 Monoclonal Antibodies to Characterize theICAM-1 Molecule

In order to determine the nature of ICAM-1, and particularly todetermine whether ICAM-1 was distinct from LFA-1, cell proteins wereimmunoprecipitated using monoclonal antibody RR1/1. Theimmunoprecipitation was performed according to the method of Rothlein,R. et al. (J. Immunol. 137:1270-1274 (1986)). JY cells were lysed at5×10⁷ cells/ml in 1% Triton X-100, 0.14 m NaCl, 10 mM Tris, pH 8.0, withfreshly added 1 mM phenylmethylsulfonylfluoride, 0.2 units per mltrypsin inhibitor aprotinin (lysis buffer) for 20 minutes at 4° C.Lysates were centrifuged at 10,000×g for 10 minutes and precleared with50 μl of a 50% suspension of CNBr-activated, glycine-quenched SepharoseC1-4B for 1 hour at 4° C. One milliliter of lysate wasimmunoprecipitated with 20 μl of a 50% suspension of monoclonal antibodyRR1/1 coupled to Sepharose C1-4B (1 mg/ml) overnight at 4° C. (Springer,T. A. et al., J. Exper. Med. 160:1901 (1984)). Sepharose-boundmonoclonal antibody was prepared using CNBr-activation of SepharoseCL-4B in carbonate buffer according to the method of March, S. et al.(Anal. Biochem. 60:149 (1974)). Washed immunoprecipitates were subjectedto SDS-PAGE and silver staining according to the procedure of Morrissey,J. H. Anal. Biochem. 117:307 (1981).

After elution of proteins with SDS sample buffer (Ho, M. K. et al., J.Biol. Chem. 258:636 (1983)) at 100° C., the samples were divided in halfand subjected to electrophoresis (SDS-8% PAGE) under reducing (FIG. 5A)or nonreducing conditions (FIG. 5B). Bands having molecular weights of50 kd and 25 kd corresponded to the heavy and light chains ofimmunoglobulins from the monoclonal antibody Sepharose (FIG. 5A, lane3). Variable amounts of other bands in the 25-50 kd weight range werealso observed, but were not seen in precipitates from hairy leukemiacells, which yielded only a 90 kd molecular weight band. The 177 kdalpha subunit and 95 kd beta subunit of LFA-1 were found to migratedifferently from ICAM-1 under both reducing (FIG. 5A, lane 2) andnonreducing (FIG. 5B, lane 2) conditions.

In order to determine the effect of monoclonal antibody RR1/1 onPHA-lymphoblast aggregation, the quantitative aggregation assaydescribed in Example 2 was employed. Thus, T cell blast cells werestimulated for 4 days with PHA, thoroughly washed, then cultured for 6days in the presence of IL-2 conditioned medium. PHA was found to beinternalized during this 6-day culture, and did not contribute to theaggregation assay. In three different assays with different T cell blastpreparations, ICAM-1 monoclonal antibodies consistently inhibitedaggregation (Table 3).

TABLE 3 Inhibition of PMA-Stimulated PHA-Lymphoblast Aggregation byRR1/1 Monoclonal Antibody^(a) % % Expt. PMA MAb AggregationInhibition^(b) 1^(c) − Control 9 — + Control 51  0 + HLA-A,B 58−14^(d ) + LFA-1 alpha 31 39 + ICAM-1 31 39 2^(e) − Control 10 — +Control 78  0 + LFA-1 beta 17 78 + ICAM-1 50 36 3^(f) − — 7 + Control70 + HLA-A,B 80 −14  + LFA-3 83 −19  + LFA-1 alpha 2 97 + LFA-1 beta 396 + ICAM-1 34 51 ^(a)Aggregation of PHA-induced lymphoblasts stimulatedwith 50 ng/ml PMA was quantitated indirectly by microscopically countingthe number of nonaggregated cells as described in Example 2. ^(b)Percentinhibition relative to cells treated with PMA and X63 monoclonalantibody. ^(c)Aggregation was measured 1 hr after the simultaneousaddition of monoclonal antibody and PMA. Cells were shaken at 175 rpm.^(d)A negative number indicates percent enhancement of aggregation.^(e)Aggregation was measured 1 hr after the simultaneous addition ofmonoclonal antibody and PMA. Cells were pelleted at 200 × G for 1 min.incubated at 37° C. for 15 min. gently resuspended, and shaken for 45min. at 100 rpm. ^(f)Cells were pretreated with PMA for 4 hr at 37° C.After monoclonal antibody was added, the tubes were incubated at 37° C.stationary for 20 min. and shaken at 75 rpm for 100 min. LFA-1monoclonal antibodies were consistently more inhibitory than ICAM-1monoclonal antibodies, whereas HLA-A, B and LFA-3 monoclonal antibodieswere without effect. These results indicate that of the monoclonalantibodies tested, only those capable of binding to LFA-1 or ICAM-1 werecapable of inhibiting cellular adhesion.

Example 10 Preparation of Monoclonal Antibody to ICAM-1 Immunization

A Balb/C mouse was immunized intraperitoneally (i.p.) with 0.5 mls of2×10⁷ JY cells in RPMI medium 103 days and 24 days prior to fusion. Onday 4 and 3 prior to fusion, mice were immunized i.p. with 10⁷ cells ofPMA differentiated U937 cells in 0.5 ml of RPMI medium.

Differentiation of U937 Cells

U937 cells (ATCC CRL-1593) were differentiated by incubating them at5×10⁵/ml in RPMI with 10% Fetal Bovine Serum, 1% glutamine and 50 pg/mlgentamycin (complete medium) containing 2 ng/ml phorbol-12-myristateacetate (PMA) in a sterile polypropylene container. On the third day ofthis incubation, one-half of the volume of medium was withdrawn andreplaced with fresh complete medium containing PMA. On day 4, cells wereremoved, washed and prepared for immunization.

Fusion

Spleen cells from the immunized mice were fused with P3×63 Ag8-653myeloma cells at a 4:1 ratio according to Galfre et al., (Nature 266:550(1977)). After the fusion, cells were plated in a 96 well flat bottomedmicrotiter plates at 105 spleen cells/well.

Selection for Anti-ICAM-1 Positive Cells

After one week, 50 μl of supernatant were screened in the qualitativeaggregation assay of Example 2 using both JY and SKW-3 as aggregatingcell lines. Cells from supernatants inhibiting JY cell aggregation butnot SKW-3 were selected and cloned 2 times utilizing limiting dilution.

This experiment resulted in the identification and cloning of threeseparate hybridoma lines which produced anti-ICAM-1 monoclonalantibodies. The antibodies produced by these hybridoma lines wereIgG_(2a), IgG_(2b), and IgM, respectively. The hybridoma cell line whichproduced the IgG_(2a) anti-ICAM-1 antibody was given the designationR6′5′D6′E9′B2. The antibody produced by the preferred hybridoma cellline was designated R6′5′D6′E9′B2 (herein referred to as “R6-5-D6”).Hybridoma cell line R6′5′D6′E9′B2 was deposited with the American TypeCulture Collection on Oct. 30, 1987 and given the designation ATCC HB9580.

Example 11 The Expression and Regulation of ICAM-1

In order to measure ICAM-1 expression, a radioimmune assay wasdeveloped. In this assay, purified RR1/1 was iodinated using iodogen toa specific activity of 10 μCi/μg. Endothelial cells were grown in 96well plates and treated as described for each experiment. The plateswere cooled at 4° C. by placing in a cold room for 0.5-1 hr, notimmediately on ice. The monolayers were washed 3× with cold completemedia and then incubated 30 m at 4° C. with ¹²⁵I RR1/1. The monolayerswere then washed 3× with complete media. The bound ¹²⁵I was releasedusing 0.1 N NaOH and counted. The specific activity of the ¹²⁵I RR1/1was adjusted using unlabeled RR1/1 to obtain a linear signal over therange of antigen densities encountered in this study. Non-specificbinding was determined in the presence of a thousand fold excess ofunlabeled RR1/1 and was subtracted from total binding to yield thespecific binding.

ICAM-1 expression, measured using the above described radioimmune assay,is increased on human umbilical vein endothelial cells (HUVEC) and humansaphenous vein endothelial cells (HSVEC) by IL-1, TNF, LPS and IFN gamma(Table 4). Saphenous vein endothelial cells were used in this study toconfirm the results from umbilical vein endothelial cells in culturedlarge vein endothelial cells derived from adult tissue. The basalexpression of ICAM-1 is 2 fold higher on saphenous vein endothelialcells than on umbilical vein endothelial cells. Exposure of umbilicalvein endothelial cell to recombinant IL-1 alpha, IL-1 beta, and TNFgamma increase ICAM-1 expression 10-20 fold. IL-1 alpha, TNF and LPSwere the most potent inducers and IL-1 was less potent on a weight basisand also at saturating concentrations for the response (Table 4). IL-1beta at 100 ng/ml increased ICAM-1 expression by 9 fold on HUVEC and 7.3fld on HSVEC with half-maximal increase occurring at 15 ng/ml. rTNF at50 ng/ml increased ICAM-1 expression 16 fold on HUVEC and 11 fold onHSVEC with half maximal effects at 0.5 ng/ml. Interferon-gamma produceda significant increase in ICAM-1 expression of 5.2 fold on HUVEC or 3.5fold on HSVEC at 10,000 U/ml. The effect of LPS at 10 μg/ml was similarin magnitude to that of rTNF. Pairwise combinations of these mediatorsresulted in additive or slightly less than additive effects on ICAM-1expression (Table 4). Cross-titration of rTNF with rIL-1 beta or rIFNgamma showed no synergism between these at suboptimal or optimalconcentrations.

Since LPS increased ICAM-1 expression on endothelial cells at levelssometimes found in culture media, the possibility that the basal ICAM-1expression might be due to LPS was examined. When several serum batchswere tested it was found that low endotoxin serum gave lower ICAM-1basal expression by 25%. All the results reported here were forendothelial cells grown in low endotoxin serum. However, inclusion ofthe LPS neutralizing antibiotic polymyxin B at 10 μg/ml decreased ICAM-1expression only an additional 25% (Table 4). The increase in ICAM-1expression on treatment with IL-1 or TNF was not effected by thepresence of 10 pg/ml polymyxin B which is consistent with the lowendotoxin levels in these preparations (Table 4).

TABLE 4 Anti-ICAM-1 Monoclonal Antibodies ¹²⁵I Specifically bound (CPM)Condition (16 hr) HUVEC HSVEC control 603 ± 11 — 1132 ± 31 — 100 ng/mlrIL-1 beta 5680 ± 633  9x  8320 ± 766  7.3x 50 ng/ml rIL-1 alpha 9910 ±538 16x — — 50 ng/ml rTNF alpha  9650 ± 1500 16x 12690 ± 657 11.2x 10μg/ml LPS 9530 ± 512 16x 10459 ± 388  9.2x 10 ng/ml rIFN gamma 3120 ±308  5.2x  4002 ± 664  3.5x rIL-1 beta + rTNF  1469 ± 1410 24x 16269 ±660 14x rIL-1 beta + LPS 13986 ± 761  23x 10870 ± 805 10x rIL-1 beta +rIFN 7849 ± 601 13x  8401 ± 390  7.4x gamma rTNF + LPS 15364 ± 1241 24x 16141 ± 1272 14x rTNF + rIFN gamma 13480 ± 1189 22x 13238 ± 761 12xLPS + IFN gamma 10206 ± 320  17x 10987 ± 668 10x polymyxin B 480 ± 23 —— — (10 μg/ml) polymyxin B + rIL-1 5390 ± 97  11x — — polymyxin B + rTNF9785 ± 389 20x — — 1 μg/ml LPS 7598 ± 432 13x — — polymyxin B + LPS 510± 44  1.1x Upregulation of ICAM-1 expression on HVEC and HSVEC-HUVEC orHSVEC were seeded into 96 well plates at 1:3 from a confluent monolayerand allowed to grow to confluence. Cells were then treated with theindicated materials or media for 16 hr and the RIA done as in methods.All points were done in quadruplicate.

Example 12 Kinetics of Interleukin 1 and Gamma Interferon Induction ofICAM-1

The kinetics of interleukin 1 and gamma interferon effects on ICAM-1expression on dermal fibroblasts were determined using the ¹²⁵I goatanti-mouse IgG binding assay of Dustin, M. L. et al. (J. Immunol.137:245-254 (1986); which reference is herein incorporated byreference). To perform this binding assay, human dermal fibroblasts weregrown in a 96-well microtiter plate to a density of 2−8×10⁴ cells/well(0.32 cm²). The cells were washed twice with RPMI 1640 mediumsupplemented as described in Example 1. The cells were additionallywashed once with Hanks Balanced Salt Solution (HBSS), 10 mM HEPES, 0.05%NaN₃ and 10% heat-inactivated fetal bovine serum. Washing with thisbinding buffer was done at 4° C. To each well was added 50 μl of theabove-described binding buffer and 50 μl of the appropriate hybridomasupernatant with X63 and W6/32 as the negative and positive controls,respectively. After incubation for 30 minutes at 4° C., with gentleagitation, the wells were washed twice with binding buffer, and thesecond antibody ¹²⁵I-goat anti-mouse IgG, was added at 50 nCi in 100 μl.The ¹²⁵I-goat anti-mouse antibody was prepared by using Iodogen (Pierce)according to the method of Fraker, P. J. et al. (Biochem. Biophys. Res.Commun. 80:849 (1978)). After 30 minutes at 4° C., the wells were washedtwice with 200 μl of binding buffer and the cell layer was solubilizedby adding 100 μl of 0.1 N NaOH. This and a 100 μl wash were counted in aBeckman 5500 gamma counter. The specific counts per minute bound wascalculated as [cpm with monoclonal antibody]-[cpm with X63]. All steps,including induction with specific reagents, were carried out inquadruplicate.

The effect of interleukin 1 with a half-life for ICAM-1 induction of 2hours was more rapid than that of gamma interferon with a half-life of3.75 hours (FIG. 6). The time course of return to resting levels ofICAM-1 appeared to depend upon the cell cycle or rate of cell growth. Inquiescent cells, interleukin 1 and gamma interferon effects are stablefor 2-3 days, whereas in log phase cultures, ICAM-1 expression is nearbaseline 2 days after the removal of these inducing agents.

The dose response curves for induction of ICAM-1 by recombinant mouseand human interleukin 1, and for recombinant human gamma interferon, areshown in FIG. 7. Gamma interferon and interleukin 1 were found to havesimilar concentration dependencies with nearly identical effects at 1ng/ml. The human and mouse recombinant interleukin 1 also have similarcurves, but are much less effective than human interleukin 1preparations in inducing ICAM-1 expression.

Cyclohexamide, an inhibitor of protein synthesis, and actinomycin D, aninhibitor of mRNA synthesis, abolish the effects of both interleukin 1and gamma interferon on ICAM-1 expression on fibroblasts (Table 5).Furthermore, tunicamycin, an inhibitor of N-linked glycosylation, onlyinhibited the interleukin 1 effect by 43%. These results indicate thatprotein and mRNA synthesis, but not N-linked glycosylation, are requiredfor interleukin 1 and gamma interferon-stimulated increases in ICAM-1expression.

TABLE 5 Effects of Cycloheximide, Actinomycin D, and Tunicamycin onICAM-1 Induction by IL 1 and gamma IFN on Human Dermal Fibroblasts^(a)¹²⁵I Goat Anti-Mouse IgG Specifically Bound (cpm) Treatment anti-ICAM-1anti-HLA-A,B,C Control (4 hr) 1524 ± 140 11928 ± 600 +cycloheximide 1513± 210 10678 ± 471 +actinomycin D 1590 ± 46  12276 ± 608 +tunicamycin1461 ± 176 12340 ± 940 IL 1 (10 U/ml) (4 hr) 4264 ± 249 12155 ± 510+cycloheximide 1619 ± 381 12676 ± 446 +actinomycin D 1613 ± 88  12294 ±123 +tunicamycin 3084 ± 113 13434 ± 661 IFN-γ (10 U/ml) (18 hr) 4659 ±109 23675 ± 500 +cycloheximide 1461 ± 59  10675 ± 800 +actinomycin D1326 ± 186 12089 ± 550 ^(a)Human fibroblasts were grown to a density of8 × 10⁴ cells/0.32 cm² well. Treatments were carried out in a finalvolume of 50 μl containing the indicated reagents. Cycloheximide,actinomycin D, and tunicamycin were added at 20 μg/ml, 10 μM, and 2μg/ml, respectively, at the same time as the cytokines. All points aremeans of quadruplicate wells ± SD.

Example 13 The Tissue Distribution of ICAM-1

Histochemical studies were performed on frozen tissue of human organs todetermine the distribution of ICAM-1 in thymus, lymph nodes, intestine,skin, kidney, and liver. To perform such an analysis, frozen tissuesections (4 μm thick) of normal human tissues were fixed in acetone for10 minutes and stained with the monoclonal antibody, RR1/1 by animmunoperoxidase technique which employed the avidin-biotin complexmethod (Vector Laboratories, Burlingame, Calif.) described byCerf-Bensussan, N. et al. (J. Immunol. 130:2615 (1983)). Afterincubation with the antibody, the sections were sequentially incubatedwith biotinylated horse anti-mouse IgG and avidin-biotinylatedperoxidase complexes. The sections were finally dipped in a solutioncontaining 3-amino-9-ethyl-carbazole (Aldrich Chemical Co., Inc.,Milwaukee, Wis.) to develop a color reaction. The sections were thenfixed in 4% formaldehyde for 5 minutes and were counterstained withhematoxylin. Controls included sections incubated with unrelatedmonoclonal antibodies instead of the RR1/1 antibody.

ICAM-1 was found to have a distribution most similar to that of themajor histocompatibility complex (MHC) Class II antigens. Most of theblood vessels (both small and large) in all tissues showed staining ofendothelial cells with ICAM-1 antibody. The vascular endothelialstaining was more intense in the interfollicular (paracortical) areas inlymph nodes, tonsils, and Peyer's patches as compared with vessels inkidney, liver, and normal skin. In the liver, the staining was mostlyrestricted to sinusoidal lining cells; the hepatocytes and theendothelial cells lining most of the portal veins and arteries were notstained.

In the thymic medulla, diffuse staining of large cells and a dendriticstaining pattern was observed. In the cortex, the staining pattern wasfocal and predominantly dendritic. Thymocytes were not stained. In theperipheral lymphoid tissue, the germinal center cells of the secondarylymphoid follicles were intensely stained. In some lymphoid follicles,the staining pattern was mostly dendritic, with no recognizable stainingof lymphocytes. Faint staining of cells in the mantle zone was alsoobserved. In addition, dendritic cells with cytoplasmic extensions(interdigitating reticulum cells) and a small number of lymphocytes inthe interfollicular or paracortical areas stained with the ICAM-1binding antibody.

Cells resembling macrophages were stained in the lymph nodes and laminapropria of the small intestine. Fibroblast-like cells (spindle-shapedcells) and dendritic cells scattered in the stroma of most of the organsstudied stained with the ICAM-1 binding antibody. No staining wasdiscerned in the Langerhans/indeterminant cells in the epidermis. Nostaining was observed in smooth muscle tissue.

The staining of epithelial cells was consistently seen in the mucosa ofthe tonsils. Although hepatocytes, bile duct epithelium, intestinalepithelial cells, and tubular epithelial cells in kidney did not stainin most circumstances, sections of normal kidney tissue obtained from anephrectomy specimen with renal cell carcinoma showed staining of manyproximal tubular cells for ICAM-1. These tubular epithelial cells alsostained with an anti-HLA-DR binding antibody.

In summary, ICAM-1 is expressed on non-hematopoietic cells such asvascular endothelial cells and on hematopoietic cells such as tissuemacrophages and mitogen-stimulated T lymphocyte blasts. ICAM-1 was foundto be expressed in low amounts on peripheral blood lymphocytes.

Example 14 The Purification of ICAM-1 by Monoclonal Antibody AffinityChromatography General Purification Scheme

ICAM-1 was purified from human cells or tissue using monoclonal antibodyaffinity chromatography. Monoclonal antibody, RR1/1, reactive withICAM-1 was first purified, and coupled to an inert column matrix. Thisantibody is described by Rothlein, R. et al. J. Immunol. 137:1270-1274(1986), and Dustin, M. L. et al. (J. Immunol. 137:245 (1986). ICAM-1 wassolubilized from cell membranes by lysing the cells in a non-ionicdetergent, Triton X-100, at a near neutral pH. The cell lysatecontaining solubilized ICAM-1 was then passed through pre-columnsdesigned to remove materials that bind nonspecifically to the columnmatrix material, and then through the monoclonal antibody column matrix,allowing the ICAM-1 to bind to the antibody. The antibody column wasthen washed with a series of detergent wash buffers of increasing pH upto pH 11.0. During these washes ICAM-1 remained bound to the antibodymatrix, while non-binding and weakly binding contaminants were removed.The bound ICAM-1 was then specifically eluted from the column byapplying a detergent buffer of pH 12.5.

Purification of Monoclonal Antibody RR1/1 and Covalent Coupling toSepharose CL-4B.

The anti-ICAM-1 monoclonal antibody RR1/1 was purified from the ascitesfluid of hybridoma-bearing mice, or from hybridoma culture supernates bystandard techniques of ammonium sulfate precipitation and protein Aaffinity chromatography (Ey et al., Immunochem. 15:429 (1978)). Thepurified IgG, or rat IgG (Sigma Chemical Co., St. Louis, Mo.) wascovalently coupled to Sepharose CL-4B (Pharmacia, Upsala, Sweden) usinga modification of the method of March et al. (Anal. Biochem. 60:149(1974)). Briefly, Sepharose CL-4B was washed in distilled water,activated with 40 mg/ml CNBr in 5 M K₂HPO₄ (pH approximately 12) for 5minutes, and then washed extensively with 0.1 mM HCl at 4° C. Thefiltered, activated Sepharose was resuspended with an equal volume ofpurified antibody (2-10 mg/ml in 0.1 M NaHCO₃, 0.1 M NaCl). Thesuspension was incubated for 18 hours at 4° C. with gentle end-over-endrotation. The supernatant was then monitored for unbound antibody byabsorbance at 280 nm, and remaining reactive sites on the activatedSepharose were saturated by adding glycine to 0.05 M. Couplingefficiency was usually greater than 90%.

Detergent Solubilization of Membranes Prepared from Human Spleen.

All procedures were done at 4° C. Frozen human spleen (200 g fragments)from patients with hairy cell leukemia were thawed on ice in 200 mlTris-saline (50 mM Tris, 0.14 M NaCl, pH 7.4 at 4° C.) containing 1 mMphenylmethylsulfonylfluoride (PMSF), 0.2 U/ml aprotinin, and 5 mMiodoacetamide. The tissue was cut into small pieces, and homogenized at4° C. with a Tekmar power homogenizer. The volume was then brought to300 ml with Tris-saline, and 100 ml of 10% Tween 40 (polyoxyethylenesorbitan monopalmitate) in Tris-saline was added to achieve a finalconcentration of 2.5% Tween 40.

To prepare membranes, the homogenate was extracted using three strokesof a Dounce or, more preferably, a Teflon Potter Elvejhem homogenizer,and then centrifuged at 1000×g for 15 minutes. The supernatant wasretained and the pellet was re-extracted with 200 ml of 2.5% Tween 40 inTris-saline. After centrifugation at 1000×g for 15 minutes, thesupernatants from both extractions were combined and centrifuged at150,000×g for 1 hour to pellet the membranes. The membranes were washedby resuspending in 200 ml Tris-saline, centrifuged at 150,000×g for 1hour. The membrane pellet was resuspended in 200 ml Tris-saline and washomogenized with a motorized homogenizer and Teflon pestle until thesuspension was uniformly turbid. The volume was then brought up to 900ml with Tris-saline, and N-lauroyl sarcosine was added to a finalconcentration of 1%. After stirring at 4° C. for 30 minutes, insolublematerial in the detergent lysate was removed by centrifugation at150,000×g for 1 hour. Triton X-100 was then added to the supernatant toa final concentration of 2%, and the lysate was stirred at 4° C. for 1hour.

Detergent Solubilization of JY B-Lymphoblastoid Cells

The EBV-transformed B-lymphoblastoid cell line JY was grown in RPMI-1640containing 10% fetal calf serum (FCS) and 10 mM HEPES to an approximatedensity of 0.8−1.0×10⁶ cells/ml. To increase the cell surface expressionof ICAM-1, phorbol 12-myristate 13-acetate (PMA) was added at 25 ng/mlfor 8-12 hours before harvesting the cells. Sodium vanadate (50 μM) wasalso added to the cultures during this time. The cells were pelleted bycentrifugation at 500×g for 10 minutes, and washed twice in Hank'sBalanced Salt Solution (HBSS) by resuspension and centrifugation. Thecells (approximately 5 g per 5 liters of culture) were lysed in 50 ml oflysis buffer (0.14 M NaCl, 50 mM Tris pH 8.0, 1% Triton X-100, 0.2 U/mlaprotinin, 1 mM PMSF, 50 μM sodium vanadate) by stirring at 4° C. for 30minutes. Unlysed nuclei and insoluble debris were removed bycentrifugation at 10,000×g for 15 minutes, followed by centrifugation ofthe supernatant at 150,000×g for 1 hour, and filtration of thesupernatant through Whatman 3 mm filter paper.

Affinity Chromatography of ICAM-1 for Structural Studies

For large scale purification of ICAM-1 to be used in structural studies,a column of 10 ml of RR1/1-Sepharose CL-4B (coupled at 2.5 mg ofantibody/ml of gel), and two 10 ml pre-columns of CNBr-activated,glycine-quenched Sepharose CL-4B, and rat-IgG coupled to Sepharose CL-4B(2 mg/ml) were used. The columns were connected in series, andpre-washed with 10 column volumes of lysis buffer, 10 column volumes ofpH 12.5 buffer (50 mM triethylamine, 0.1% Triton X-100, pH 12.5 at 4°C.), followed by equilibration with 10 column volumes of lysis buffer.One liter of the detergent lysate of human spleen was loaded at a flowrate of 0.5-1.0 ml per minute. The two pre-columns were used to removenon-specifically binding material from the lysate before passage throughthe RR1/1-Sepharose column.

After loading, the column of RR1/1-Sepharose and bound ICAM-1 was washedsequentially at a flow rate of 1 ml/minute with a minimum of 5 columnvolumes each of the following: 1) lysis buffer, 2) 20 mM Tris pH8.0/0.14 M NaCl/0.1% Triton X-100, 3) 20 mM glycine pH 10.0/0.1% TritonX-100, and 4) 50 mM triethylamine pH 11.0/0.1% Triton X-100. All washbuffers contained 1 mM PMSF and 0.2 U/ml aprotinin. After washing, theremaining bound ICAM-1 was eluted with 5 column volumes of elutionbuffer (50 mM triethylamine/0.1% Triton X-100/pH 12.5 at 4° C.) at aflow rate of 1 ml/3 minutes. The eluted ICAM-1 was collected in 1 mlfractions and immediately neutralized by the addition of 0.1 ml of 1 MTris, pH 6.7. Fractions containing ICAM-1 were identified bySDS-polyacrylamide electrophoresis of 10 μl aliquots (Springer et al.,J. Exp. Med. 160:1901 (1984)), followed by silver staining (Morrissey,J. H., Anal. Biochem. 117:307 (1981)). Under these conditions, the bulkof the ICAM-1 eluted in approximately 1 column volume and was greaterthan 90% pure as judged from silver-stained electropherograms (a smallamount of IgG, leeched from the affinity matrix, was the majorcontaminant). The fractions containing ICAM-1 were pooled andconcentrated approximately 20-fold using Centricon-30 microconcentrators(Amicon, Danvers, M A). The purified ICAM-1 was quantitated by Lowryprotein assay of an ethanol-precipitated aliquot of the pool:approximately 500 μg of pure ICAM-1 were produced from the 200 g ofhuman spleen.

Approximately 200 pg of purified ICAM-1 was subjected to a second stagepurification by preparative SDS-polyacrylamide gel electrophoresis. Theband representing ICAM-1 was visualized by soaking the gel in 1 M KCl.The gel region which contained ICAM-1 was then excised and electroelutedaccording to the method of Hunkapiller et al., Meth. Enzymol. 91:227-236(1983). The purified protein was greater than 98% pure as judged bySDS-PAGE and silver staining.

Affinity Purification of ICAM-1 for Functional Studies

ICAM-1 for use in functional studies was purified from detergent lysatesof JY cells as described above, but on a smaller scale (a 1 ml column ofRR1/1-Sepharose), and with the following modifications. All solutionscontained 50 μM sodium vanadate. After washing the column with pH 11.0buffer containing 0.1% Triton X-100, the column was washed again withfive column volumes of the same buffer containing 1%n-octyl-beta-D-glucopyranoside (octylglucoside) in place of 0.1% TritonX-100. The octylglucoside detergent displaces the Triton X-100 bound tothe ICAM-1, and unlike Triton X-100, can be subsequently removed bydialysis. The ICAM-1 was then eluted with pH 12.5 buffer containing 1%octylglucoside in place of 0.1% Triton X-100, and was analyzed andconcentrated as described above.

Example 15 Characteristics of Purified ICAM-1

ICAM-1 purified from human spleen migrates in SDS-polyacrylamide gels asa broad band of M_(r) of 72,000 to 91,000. ICAM-1 purified from JY cellsalso migrates as a broad band of M_(r) of 76,500 to 97,000. These M_(r)are within the reported range for ICAM-1 immunoprecipitated fromdifferent cell sources: M_(r)=90,000 for JY cells, 114,000 on themyelomonocytic cell line U937, and 97,000 on fibroblasts (Dustin et al.,J. Immunol. 137:245 (1986)). This wide range in M_(r) has beenattributed to an extensive, but variable degree of glycosylation. Thenon-glycosylated precursor has a M_(r) of 55,000 (Dustin et al.). Theprotein purified from either JY cells or human spleen retains itsantigenic activity as evidenced by its ability to re-bind to theoriginal affinity column, and by immunoprecipitation withRR1/1-Sepharose and SDS-polyacrylamide electrophoresis.

To produce peptide fragments of ICAM-1, approximately 200 μg was reducedwith 2 mM dithiothreitol/2% SDS, followed by alkylation with 5 mMiodoacetic acid. The protein was precipitated with ethanol, andredissolved in 0.1 M NH₄CO₃/0.1 mM CaCl₂/0.1% zwittergent 3-14(Calbiochem), and digested with 1% w/w trypsin at 37° C. for 4 hours,followed by an additional digestion with 1% trypsin for 12 hours at 37°C. The tryptic peptides were purified by reverse-phase HPLC using a0.4×15 cm C4 column (Vydac). The peptides were eluted with a lineargradient of 0% to 60% acetonitrile in 0.1% trifluoroacetic acid.Selected peptides were subjected to sequence analysis on a gas phasemicrosequenator (Applied Biosystems). The sequence information obtainedfrom this study is shown in Table 6.

TABLE 6 Amino Acid Sequences of ICAM-1 Tryptic Peptides Amino AcidPeptide Residue 50a 50b 46a 46b X 45 K AA J U 0 M1 1 [T/V] A (V/A) E V SL E A L V L 2 F S Q P E F N L G L T L/E 3 L I T A L P P D S G L P/(G) 4T S F A A T L V I P 5 V L P P P V R L E G/Y 6 Y G L L N T P V T N/L 7 PW P P V Y Q T P (N) 8 T P I I T/I G G C P/V (Q) 9 S F G (G) L — L S K(E) 10 E E (Q) — D E T (D) 11 A S D/P K S L S 12 G/S V V P F F C 13 A TD Q S E D 14 G V W V/L A — Q 15 I K T P 16 S K 17 A 18 P 19 X 20 Q 21 L( ) = Low confidence sequence. [ ] = Very low confidence sequence. / =Indicates ambiguity in the sequence; most probable amino acid is listedfirst. a = Major peptide. b = Minor peptide.

Example 16 Cloning of the ICAM-1 Gene

The gene for ICAM-1 may be cloned using any of a variety of procedures.For example, the amino acid sequence information obtained through thesequencing of the tryptic fragments of ICAM-1 (Table 6) can be used toidentify an oligonucleotide sequence which would correspond to theICAM-1 gene. Alternatively, the ICAM-1 gene can be cloned usinganti-ICAM-1 antibody to detect clones which produce ICAM-1.

Cloning of the Gene for ICAM-1 Through the Use of Oligonucleotide Probes

Using the genetic code (Watson, J. D., In: Molecular Biology of theGene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977)), one ormore different oligonucleotides can be identified, each of which wouldbe capable of encoding the ICAM-1 tryptic peptides. The probability thata particular oligonucleotide will, in fact, constitute the actual ICAM-1encoding sequence can be estimated by considering abnormal base pairingrelationships and the frequency with which a particular codon isactually used (to encode a particular amino acid) in eukaryotic cells.Such “codon usage rules” are disclosed by Lathe, R., et-al., J. Molec.Biol. 183:1-12 (1985). Using the “codon usage rules” of Lathe, a singleoligonucleotide, or a set of oligonucleotides, that contains atheoretical “most probable” nucleotide sequence (i.e. the nucleotidesequence having the lowest redundancy) capable of encoding the ICAM-1tryptic peptide sequences is identified.

The oligonucleotide, or set of oligonucleotides, containing thetheoretical “most probable” sequence capable of encoding the ICAM-1fragments is used to identify the sequence of a complementaryoligonucleotide or set of oligonucleotides which is capable ofhybridizing to the “most probably” sequence, or set of sequences. Anoligonucleotide containing such a complementary sequence can be employedas a probe to identify and isolate the ICAM-1 gene (Maniatis, T., etal., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1982).

As described in Section C, supra, it is possible to clone the ICAM-1gene from eukaryotic DNA preparations suspected of containing this gene.To identify and clone the gene which encodes the ICAM-1 protein, a DNAlibrary is screened for its ability to hybridize with theoligonucleotide probes described above. Because it is likely that therewill be only two copies of the gene for ICAM-1 in a normal diploid cell,and because it is possible that the ICAM-1 gene may have largenon-transcribed intervening sequences (introns) whose cloning is notdesired, it is preferable to isolate ICAM-1-encoding sequences from acDNA library prepared from the mRNA of an ICAM-1-producing cell, ratherthan from genomic DNA. Suitable DNA, or cDNA preparations areenzymatically cleaved, or randomly sheared, and ligated into recombinantvectors. The ability of these recombinant vectors to hybridize to theabove-described oligonucleotide probes is then measured. Procedures forhybridization are disclosed, for example, in Maniatis, T., MolecularCloning A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1982) or in Haymes, B. T., et al., Nucleic AcidHybridization a Practical Approach, IRL Press, Oxford, England (1985).Vectors found capable of such hybridization are then analyzed todetermine the extent and nature of the ICAM-1 sequences which theycontain. Based purely on statistical considerations, a gene such as thatwhich encodes the ICAM-1 molecule could be unambiguously identified (viahybridization screening) using an oligonucleotide probe having only 18nucleotides.

Thus, in summary, the actual identification of ICAM-1 peptide sequencespermits the identification of a theoretical “most probable” DNAsequence, or a set of such sequences, capable of encoding such apeptide. By constructing an oligonucleotide complementary to thistheoretical sequence (or by constructing a set of oligonucleotidescomplementary to the set of “most probable” oligonucleotides), oneobtains a DNA molecule (or set of DNA molecules), capable of functioningas a probe to identify and isolate the ICAM-1 gene.

Using the ICAM-1 peptide sequences of Table 6, the sequence of the “mostprobable” sequence of an oligonucleotide capable of encoding the AA andJ peptides was determined (Tables 7 and 8, respectively).Oligonucleotides complementary to these sequences were synthesized andpurified for use as probes to isolate ICAM-1 gene sequences. Suitablesize-selected cDNA libraries were generated from the poly(A)⁺ RNA ofboth PMA-induced HL-60 cells and from PS-stimulated umbilical veinendothelial cells. A size-selected cDNA library was prepared usingpoly(A)⁺ RNA from PMA-induced HL-60 cells according to the method ofGubler, U., et al. ((Gene 25:263-269 (1983)) and Corbi, A., et al. (EMBOJ. 6:4023-4028 (1987)), which references are herein incorporated byreference.

A size-selected cDNA library was prepared using poly(A)⁺ RNA fromumbilical vein endothelial cells which had been stimulated for 4 hourswith PS 5 μg/ml. The RNA was extracted by homogenizing the cells in 4 Mguanidinium isothiocyanate and subjecting the supernatant toultracentrifugation through a CsCl gradient (Chirgwin, J. M., et al.,Biochem. 18:5294-5299 (1979)). Poly(A)⁺ RNA was isolated from themixture of total RNA species through the use of oligo (dT)-cellulosechromatography (Type 3, Collaborative Research) (Aviv, H., et al., Proc.Natl. Acad. Sci. (USA) 69:1408-1412 (1972).

TABLE 7 Oligonucleotide Complementary to the Most Probable NucleotideSequence Capable of Encoding the ICAM-1 AA Peptide Amino Acid MostProbable Residue of ICAM-1 Sequence Encoding Complementary ICAM-1 AAPeptide AA Peptide Sequence 5′ 3′ 162 Glu G C A T G C 163 Leu C G T A GC 164 Asp G C A T C G 165 Leu C G T A G C 166 Arg C G G C G C 167 Pro CG C G C G 168 Gln C G A T G C 169 Gly G C G C C G 170 Leu C G T A G C171 Glu G C A T G C 172 Leu C G T A G C 173 Phe T A T A T A 174 Glu G CA T G C 3′ 5′ 175 Asn A T A T C G 176 Thr A T C G C G 177 Ser U A C G A3′ 5′

TABLE 8 Oligonucleotide Complementary to the Most Probable NucleotideSequence Capable of Encoding the ICAM-1 J Peptide Amino Acid MostProbable Residue of ICAM-1 Sequence Encoding Complementary ICAM-1 AAPeptide AA Peptide Sequence 5′ 3′ 19 Val G C T A G C 20 Thr A T C G C G21 Cys T A G C C G 22 Ser T A C G C G 23 Thr A T C G C G 24 Ser T A C GC G 25 Cys T A G C T A 26 Asp G C A T C G 27 Gln C G A T G C 28 Pro C GC G C G 29 Lys A T A T 3′ 5′

First strand cDNA was synthesized using 8 μg of poly(A)⁺ RNA, avianmyeloblastosis virus reverse transcriptase (Life Sciences) and anoligo(dT) primer. The DNA-RNA hybrid was digested with RNase H (BRL) andthe second strand was synthesized using DNA polymerase I (New EnglandBiolabs). The product was methylated with Eco R1 methylase (New EnglandBiolabs), blunt end ligated to Eco R1 linkers (New England Biolabs),digested with Eco R1 and size selected on a low melting point agarosegel. cDNA greater than 500 bp were ligated to λgt10 which had previouslybeen Eco R1 digested and dephosphorylated (Stratagene) The product ofthe ligation was then packaged (Stratagene gold).

The umbilical vein endothelial cell and HL-60 cDNA libraries were thenplated at 20,000 PFU/150 mm plate. Recombinant DNA was transferred induplicate to nitrocellulose filters, denatured in 0.5 M NaOH/1.5M NaCl,neutralized in 1M Tris, pH7.5/1.5M NaCl and baked at 80° C. for 2 hours(Benton, W. D., et al., Science 196:180-182 (1977)). Filters wereprehybridized and hybridized in 5×SSC containing 5×Denhardt's solution,50 mM NaPO₄ and 1 pg/ml salmon sperm DNA. Prehybridization was carriedout at 45′ for 1 hour.

Hybridization was carried out using 32 bp(′5-TTGGGCTGGTCACAGGAGGTGGAGCAGGTGAC) or 47 bp(5′-GAGGTGTTCTCAAACAGCTCCAGGCCCTGG GGCCGCAGGTCCAGCTC) anti-senseoligonucleotides based, in the manner discussed above, on the ICAM-1tryptic peptides J and AA, respectively (Tables 7 and 8) (Lathe, R., J.Molec. Biol., 183:1-12 (1985)). Oligonucleotides were end labeled withγ-(³²P)ATP using T4 polynucleotide kinase and conditions recommended bythe manufacturer (New England Biolabs). Following overnighthybridization the filters were washed twice with 2×SSC/0.1% SDS for 30minutes at 45° C. Phages were isolated from those plaques whichexhibited hybridization, and were purified by successive replating andrescreening.

Cloning of the Gene for ICAM-1 Through the Use of Anti-ICAM-1 Antibody

The gene for ICAM-1 may alternatively be cloned through the use ofanti-ICAM-1 antibody. DNA, or more preferably cDNA, is extracted andpurified from a cell which is capable of expressing ICAM-1. The purifiedcDNA is fragmentized (by shearing, endonuclease digestion, etc.) toproduct a pool of DNA or cDNA fragments. DNA or cDNA fragments from thispool are then cloned into an expression vector in order to produce agenomic library of expression vectors whose members each contain aunique cloned DNA or cDNA fragment.

Example 17 Analysis of the cDNA Clones

Phage DNA from positive clones were digested with Eco R1 and examined bySouthern analysis using a cDNA from one clone as a probe. Maximal sizecDNA inserts which cross-hybridized were subcloned into the Eco R1 siteof plasmid vector pGEM 4Z (Promega). HL-60 subclones containing the cDNAin both orientations were deleted by exonuclease III digestion(Henikoff, S., Gene 28:351-359 (1984)) according to the manufacturersrecommendations (Erase-a-Base, Promega). Progressively deleted cDNAswere then cloned and subjected to dideoxynucleotide chain terminationsequencing (Sanger, F. et al., Proc. Natl. Acad. Sci. (USA) 74:5463-5467(1977)) according to the manufacturers recommendations (Sequence, U.S.Biochemical). The HL-60 cDNA 5′ and coding regions were sequencedcompletely on both strands and the 3′ region was sequenced approximately70% on both strands. A representative endothelial cDNA was sequencedover most of its length by shotgun cloning of 4 bp-recognitionrestriction enzyme fragments.

The cDNA sequence of one HL-60 and one endothelial cell cDNA wasestablished (FIG. 8). The 3023 bp sequence contains a short 5′untranslated region and a 1.3 kb 3′ untranslated region with a consensuspolyadenylation signal at position 2966. The longest open reading framebegins with the first ATG at position 58 and ends with a TGA terminatingtriplet at position 1653. Identity between the translated amino acidsequence and sequences determined from 8 different tryptic peptidestotaling 91 amino acids (underlined in FIG. 8) confirmed that authenticICAM-1 cDNA clones had been isolated. The amino acid sequences of ICAM-1tryptic peptides are shown in Table 9.

TABLE 9 Amino Acid Sequences of ICAM-1 Tryptic Peptides Pep- tideResidues Sequence J 14-29 X G S V L V T C S T S C D Q P K U 30-39 L L GI E T P L (P) (K) 50 78-85   (T) F L T V Y X T X 89-95   V E L A P L PAA 161-182   X E L D L R P Q G L E  -- L F E X T S A P X Q L K 232-246 LN P T V T Y G X D S F S A K 45 282-295 S F P A P N V (T/I) L X K P Q(V/L) -- Indicates that the sequence continues on the next line.Underlined sequences were used to prepare oligonucleotide probes.

Hydrophobicity analysis (Kyte, J., et al., J. Molec. Biol., 157:105-132(1982)) suggests the presence of a 27 residue signal sequence. Theassignment of the +1 glutamine is consistent with our inability toobtain N-terminal sequence on 3 different ICAM-1 protein preparations;glutamine may cyclize to pyroglumatic acid, resulting in a blockedN-terminus. The translated sequence from 1 to 453 is predominantlyhydrophilic followed by a 24 residue hydrophobic putative transmembranedomain. The transmembrane domain is immediately followed by severalcharged residues contained within a 27 residue putative cytoplasmicdomain.

The predicted size of the mature polypeptide chain is 55,219 daltons, inexcellent agreement with the observed size of 55,000 for deglycosylatedICAM-1 (Dustin, M. L., et al., J. Immunol. 137:245-254 (1986)). EightN-linked glycosylation sites are predicted. Absence of asparagine in thetryptic peptide sequences of 2 of these sites confirm theirglycosylation and their extracellular orientation. Assuming 2,500daltons per high mannose N-linked carbohydrate, a size of 75,000 daltonsis predicted for the ICAM-1 precursor, compared to the observed six of73,000 daltons (Dustin, M. L., et al., J. Immunol. 137:245-254 (1986)).After conversion of high mannose to complex carbohydrate, the matureICAM-1 glycoprotein is 76 to 114 kd, depending on cell type (Dustin, M.L., et al., J. Immunol. 137:245-254 (1986)). Thus ICAM-1 is a heavilyglycosylated but otherwise typical integral membrane protein.

Example 18 ICAM-1 is an Integrin-Binding Member of the ImmunoglobulinSupergene Family

Alignment of ICAM-1 internal repeats was performed using the Microgenieprotein alignment program (Queen, C., et al., Nucl. Acid Res.,12:581-599 (1984)) followed by inspection. Alignment of ICAM-1 to IgM,N-CAM and MAG was carried out using Microgenie and the ALIGN program(Dayhoff, M. O., et al., Meth. Enzymol. 91:524-545 (1983)). Four proteinsequence databases, maintained by the National Biomedical ResearchFoundation, were searched for protein sequence similarities using theFASTP program of Williams and Pearson (Lipman, D. J., et al., Science227:1435-1439 (1985)).

Since ICAM-1 is a ligand of an integrin, it was unexpected that it wouldbe a member of the immunoglobulin supergene family. However, inspectionof the ICAM-1 sequence shows that it fulfills all criteria proposed formembership in the immunoglobulin supergene family. These criteria arediscussed in turn below.

The entire extracellular domain of ICAM-1 is constructed from 5homologous immunoglobulin-like domains which are shown aligned in FIG.9A. Domains 1-4 are 88, 97, 99, and 99 residues long, respectively andthus are of typical Ig domain size; domain 5 is truncated within 68residues. Searches of the NBRF data base using the FASTP programrevealed significant homologies with members of the immunoglobulinsupergene family including IgM and IgG C domains, T cell receptor αsubunit variable domain, and alpha 1 beta glycoprotein (FIG. 9B-D).

Using the above information, the amino acid sequence of ICAM-1 wascompared with the amino acid sequences of other members of theimmunoglobulin supergene family.

Three types of Ig superfamily domains, V, C1, and C2 have beendifferentiated. Both V and C domains are constructed from 2 β-sheetslinked together by the intradomain disulfide bond; V domains contain 9anti-parallel β-strands while C domains have 7. Constant domains weredivided into the C1- and C2-sets based on characteristic residues shownin FIG. 9A. The C1-set includes proteins involved in antigenrecognition. The C2-set includes several Fc receptors and proteinsinvolved in call adhesion including CD2, LFA-3, MAG, and NCAM. ICAM-1domains were found to be most strongly homologous with domains of theC2-set placing ICAM-1 in this set; this is reflected in strongersimilarity to conserved residues in C2 than C1 domains as shown forβ-strands B—F in FIG. 9. Also, ICAM-1 domains aligned much better withβ-strands A and G of C2 domains than with these strands in V and C1domains, allowing good alignments across the entire C2 domain strength.Alignments with C2 domains from NCAM, MAG, and alpha 1-β glycoproteinare shown in FIGS. 9B and 9C; identity ranged from 28 to 33%. Alignmentswith a T cell receptor Vα 27% identity and IgM C domain 3 34% identityare also shown (FIGS. 9B, 9D).

One of the most important characteristics of immunoglobulin domains isthe disulfide-bonded cysteines bridging the B and F β strands whichstabilizes the p sheet sandwich; in ICAM-1 the cysteines are conservedin all cases except in strand f of domain 4 where a leucine is foundwhich may face into the sandwich and stabilize the contact as proposedfor some other V- and C2-sets domains. The distance between thecysteines (43, 50, 52, and 37 residues) is as described for the C2-set.

To test for the presence of intrachain disulfide bonds in ICAM-1,endothelial cell ICAM-1 was subjected to SDS-PAGE under reducing andnon-reducing conditions. Endothelial cell ICAM-1 was used because itshows less glycosylation heterogeneity than JY or hairy cell splenicICAM-1 and allows greater sensitivity to shifts in M_(r). ICAM-1 was,therefore, purified from 16 hour LPS (5 μg/ml) stimulated umbilical veinendothelial cell cultures by immunoaffinity chromatography as describedabove. Acetone precipitated ICAM-1 was resuspended in sample buffer(Laemmli, U. K., Nature 227:680-685 (1970)) with 0.25% 2-mercaptoethanolor 25 mM iodoacetamide and brought to 100° C. for 5 min. The sampleswere subjected to SDS-PAGE 4670 and silver staining 4613. Endothelialcell ICAM-1 had an apparent M_(r) of 100 Kd under reducing conditionsand 96 Kd under non-reducing conditions strongly suggesting the presenceof intrachain disulfides in native ICAM-1.

Use of the primary sequence to predict secondary structure (Chou, P. Y.,et al., Biochem. 13:211-245 (1974)) showed the 7 expected β-strands ineach ICAM-1 domain, labeled a-g in FIG. 9A upper, exactly fulfilling theprediction for an immunoglobulin domain and corresponding to thepositions of strands A-H in immunoglobulins (FIG. 9A, lower). Domain 5lacks the A and C strands but since these form edges of the sheets thesheets could still form, perhaps with strand D taking the place ofstrand C as proposed for some other C2 domains; and the characteristicdisulfide bond between the B and F strands would be unaffected. Thus,the criteria for domain size, sequence homology, conserve cysteinesforming the putative intradomain disulfide bond, presence of disulfidebonds, and predicted β sheet structure are all met for inclusion ofICAM-1 in the immunoglobulin supergene family.

ICAM-1 was found to be most strongly homologous with the NCAM and MAGglycoproteins of the C2 set. This is of particular interest since bothNCAM and MAG mediate cell-cell adhesion. NCAM is important inneuron-neuron and neuro-muscular interactions (Cunningham, B. A., etal., Science 236:799-806 (1987)), while MAG is important inneuron-oligodendrocyte and oligodendrocyte-oligodendrocyte interactionsduring myelination (Poltorak, M., et al., J. Cell Biol. 105:1893-1899(1987)). The cell surface expression of NCAM and MAG is developmentallyregulated during nervous system formation and myelination, respectively,in analogy to the regulated induction of ICAM-1 in inflammation(Springer, T. A., et al., Ann. Rev. Immunol. 5:223-252 (1987)). ICAM-1,NCAM (Cunningham, B. A., et al., Science 236:799-806 (1987)), and MAG(Salzer, J. L., et al., J. Cell. Biol. 104:957-965 (1987)) are similarin overall structure as well as homologous, since each is an integralmembrane glycoprotein constructed from 5 C2 domains forming theN-terminal extracellular region, although in NCAM some additionalnon-Ig-like sequence is present between the last C2 domain and thetransmembrane domain. ICAM-1 aligns over its entire length including thetransmembrane and cytoplasmic domains with MAG with 21% identity; thesame % identity is found comparing the 5 domains of ICAM-1 and NCAM-1. Adiagrammatic comparison of the secondary structures of ICAM-1 and MAG isshown in FIG. 10. Domain by domain comparisons show that the level ofhomology between domains within the ICAM-1 and NCAM molecules (x+s.d.21±2.8% and 18.6±3.8%, respectively) is the same as the level ofhomology comparing ICAM-1 domains to NCAM and MAG domains (20.4±3.7 and21.9±2.7, respectively). Although there is evidence for alternativesplicing in the C-terminal regions of NCAM (Cunningham, B. A., et al.,Science 236:799-806 (1987); Barthels, D., et al., EMBO J. 6:907-914(1987)) and MAG (Lai, C., et al., Proc. Natl. Acad. Sci. (USA)84:4377-4341 (1987)), no evidence for this has been found in thesequencing of endothelial or HL-60 ICAM-1 clones or in studies on theICAM-1 protein backbone and precursor in a variety of cell types(Dustin, M. L., et al., J. Immunol. 137:245-254 (1986)).

ICAM-1 functions as a ligand for LFA-1 in lymphocyte interactions with anumber of different cell types. Lymphocytes bind to ICAM-1 incorporatedin artificial membrane bilayers, and this requires LFA-1 on thelymphocyte, directly demonstrating LFA-1 interaction with ICAM-1(Marlin, S. D., et al., cell 51:813-819 (1987)). LFA-1 is a leukocyteintegrin and has no immunoglobulin-like features. Leukocyte integrinscomprise one integrin subfamily. The other two subfamilies mediatecell-matrix interactions and recognize the sequence RGD within theirligands which include fibronectin, vitronectin, collagen, and fibrinogen(Hynes, R. O., Cell 48:549-554 (1987); Ruoslahti, E., et al., Science238:491-497 (1987)). The leukocyte integrins are only expressed onleukocytes, are involved in cell-cell interactions, and the only knownligands are ICAM-1 and iC3b, a fragment of the complement component C3which shows no immunoglobulin-like features and is recognized by Mac-1(Kishimoto, T. K., et al., In: Leukocyte Typing III, McMichael, M.(ed.), Springer-Verlag, New York (1987); Springer, T. A., et al., Ann.Rev. Immunol. 5:223-252 (1987); Anderson, D. C., et al., Ann. Rev. Med.38:175-194 (1987)). Based upon sequence analysis, possible peptideswithin the ICAM-1 sequence recognized by LFA-1 are shown in Table 10.

TABLE 10 Peptides Within the ICAM-1 Sequence Possibly Recognized byLFA-1 -L-R-G-E-K-E-L- -R-G-E-K-E-L-K-R-E-P--L-R-G-E-K-E-L-K-R-E-P-A-V-G-E-P-A-E- -P-R-G-G-S- -P-G-N-N-R-K--Q-E-D-S-Q-P-M- -T-P-E-R-V-E-L-A-P-L-P-S- -R-R-D-H-H-G-A-N-F-S--D-L-R-P-Q-G-L-E-

ICAM-1 is the first example of a member of the immunoglobulin supergenefamily which binds to an integrin. Although both of these families playan important role in cell adhesion, interaction between them had notpreviously been expected. In contrast, interactions within theimmunoglobulin gene superfamily are quite common. It is quite possiblethat further examples of interactions between the integrin andimmunoglobulin families will be found. LFA-1 recognizes a liganddistinct from ICAM-1 (Springer, T. A., et al., Ann. Rev. Immunol.5:223-252 (1987)), and the leukocyte integrin Mac-1 recognizes a liganddistinct from C3bi in neutrophil-neutrophil adhesion (Anderson, D. C.,et al., Ann. Rev. Med. 38:175-194 (1987)). Furthermore, purifiedMAG-containing vesicles bind to neurites which are MAG, and thus MAGmust be capable of heterophilic interaction with a distinct receptor(Poltorak, M., et al., J. Cell Biol. 105:1893-1899 (1987)).

NCAM's role in neural-neural and neural-muscular cell interactions hasbeen suggested to be due to homophilic NCAM-NCAM interactions(Cunningham, B. A., et al., Science 236:799-806 (1987)). The importantrole of MAG in interactions between adjacent turning loops of Schwanncells enveloping axons during myelin sheath formation might be due tointeraction with a distinct receptor, or due to homophilic MAG-MAGinteractions. The homology with NCAM and the frequent occurrence ofdomain-domain interactions within the immunoglobulin supergene familyraises the possibility that ICAM-1 could engage in homophilicinteractions as well as ICAM-1-LFA-1 heterophilic interactions. However,binding of B lymphoblast cells which co-express similar densities ofLFA-1 and ICAM-1 to ICAM-1 in artificial or cellular monolayers can becompletely inhibited by pretreatment of the B lymphoblast with LFA-1MAb, while adherence is unaffected by B lymphoblast pretreatment withICAM-1 MAb. Pretreatment of the monolayer with ICAM-1 Mab completelyabolishes binding (Dustin, M. L., et al., J. Immunol. 137:245-254(1986); Marlin, S. D., et al., cell 51:813-819 (1987)). These findingsshow that if ICAM-1 homophilic interactions occur at all, they must bemuch weaker than heterophilic interaction with LFA-1.

The possibility that the leukocyte integrins recognize ligands in afundamentally different way is consistent with the presence of a 180residue sequence in their α subunits which may be important in ligandbinding and which is not present in the RGD-recognizing integrins(Corbi, A., et al. (EMBO J. 6:4023-4028 (1987)). Although Mac-1 has beenproposed to recognize RGD sequence present in iC3b 5086, there is no RGDsequence in ICAM-1 (FIG. 8). This is in agreement with the failure ofthe fibronectin peptide GRGDSP and the control peptide GRGESP to inhibitICAM-1-LFA-1 adhesion (Marlin, S. D., et al., cell 51:813-819 (1987)).However, related sequences such as PRGGS and RGEKE are present in ICAM-1in regions predicted to loop between 0 strands a and b of domain 2 and cand d of domain 2, respectively (FIG. 9), and thus may be accessible forrecognition. It is of interest that the homologous MAG molecule containsan RGD sequence between domains 1 and 2 (Poltorak, M., et al., J. CellBiol. 105:1893-1899 (1987); Salzer, J. L., et al., J. Cell. Biol.104:957-965 (1987)).

Example 19 Southern and Northern Blots

Southern blots were performed using a 5 pg of genomic DNA extracted fromthree cell lines: BL2, a Burkitt lymphoma cell line (a gift from Dr.Gilbert Lenoir); JY and Er-LCL, EBV transformed B-lymphoblastoid celllines.

The DNAs were digested with 5× the manufacturers recommended quantity ofBam H1 and Eco R1 endonucleases (New England Biolabs). Followingelectrophoresis through a 0.8% agarose gel, the DNAs were transferred toa nylon membrane (Zeta Probe, BioRad). The filter was prehybridized andhybridized following standard procedures using ICAM cDNA from HL-60labeled with α-(³²P)d XTP's by random priming (Boehringer Mannheim).Northern blots were performed using 20 pg of total RNA or 6 μg ofpoly(A)⁺ RNA. RNA was denatured and electrophoresed through a 1%agarose-formaldehyde gel and electrotransferred to Zeta Probe. Filterswere prehybridized and hybridized as described previously (Staunton, D.E., et al. Embo J. 6:3695-3701 (1987)) using the HL-60 cDNA probe of³²P-labeled oligonucleotide probes (described above).

The Southern blots using the 3 kb cDNA probe and genomic DNA digestedwith Bam H1 and Eco R1 showed single predominant hybridizing fragmentsof 20 and 8 kb, respectively, suggesting a single gene and suggestingthat most of the coding information is present within 8 kb. In blots of3 different cell lines there is no evidence of restriction fragmentpolymorphism.

Example 20 Expression of the ICAM-1 Gene

An “expression vector” is a vector which (due to the presence ofappropriate transcriptional and/or translational control sequences) iscapable of expressing a DNA (or cDNA) molecule which has been clonedinto the vector and of thereby producing a polypeptide or protein.Expression of the cloned sequences occurs when the expression vector isintroduced into an appropriate host cell. If a prokaryotic expressionvector is employed, then the appropriate host cell would be anyprokaryotic cell capable of expressing the cloned sequences. Similarly,if a eukaryotic expression vector is employed, then the appropriate hostcell would be any eukaryotic cell capable of expressing the clonedsequences. Importantly, since eukaryotic DNA may contain interveningsequences, and since such sequences cannot be correctly processed inprokaryotic cells, it is preferable to employ cDNA from a cell which iscapable of expressing ICAM-1 in order to produce a prokaryotic genomicexpression vector library. Procedures for preparing cDNA and forproducing a genomic library are disclosed by Maniatis, T., et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1982)).

The above-described expression vector genomic library is used to createa bank of host cells (each of which contains one member of the library).The expression vector may be introduced into the host cell by any of avariety of means (i.e., transformation, transfection, protoplast fusion,electroporation, etc.). The bank of expression vector-containing cellsis clonally propagated, and its members are individually assayed (usingan immunoassay) to determine whether they produce a protein capable ofbinding to anti-ICAM-1 antibody.

The expression vectors of those cells which produce a protein capable ofbinding to anti-ICAM-1 antibody are then further analyzed to determinewhether they express (and thus contain) the entire ICAM-1 gene, whetherthey express (and contain) only a fragment of the ICAM-1gene, or whetherthey express (and contain) a gene whose product, though immunologicallyrelated to ICAM-1, is not ICAM-1. Although such an analysis may beperformed by any convenient means, it is preferable to determine thenucleotide sequence of the DNA or cDNA fragment which had been clonedinto the expression vector. Such nucleotide sequences are then examinedto determine whether they are capable of encoding polypeptides havingthe same amino acid sequence as the tryptic digestion fragments ofICAM-1 (Table 6).

An expression vector which contains a DNA or cDNA molecule which encodesthe ICAM-1 gene may, thus, be recognized by: (i) the ability to directthe expression of a protein which is capable of binding to anti-ICAM-1antibody; and (ii) the presence of a nucleotide sequence which iscapable of encoding each of the tryptic fragments of ICAM-1. The clonedDNA molecule of such an expression vector may be removed from theexpression vector and isolated in pure form.

Example 21 Functional Activities of Purified ICAM-1

In cells, ICAM-1 normally functions as a surface protein associated withthe cell membrane. Therefore, the function of purified ICAM-1 was testedafter the molecule was reconstituted into artificial lipid membranes(liposomes, or vesicles) by dissolving the protein indetergent-solubilized lipids, followed by the removal of the detergentby dialysis. ICAM-1 purified from JY cells and eluted in the detergentoctylglucoside as described above was reconstituted into vesicles, andthe ICAM-1 containing vesicles were fused to glass coverslips or plasticculture wells to allow the detection of cells binding to the protein.

Preparation of Planar Membranes and Plastic-Bound Vesicles

Vesicles were prepared by the method of Gay et al. (J. Immunol. 136:2026(1986)). Briefly, egg phosphatidylcholine and cholesterol were dissolvedin chloroform and mixed in a molar ratio of 7:2. The lipid mixture wasdried to a thin film while rotating under a stream of nitrogen gas, andwas then lyophilized for 1 hour to remove all traces of chloroform. Thelipid film was then dissolved in 1% octylglucoside/0.14 M NaCl/20 mMTris (pH 7.2) to a final concentration of phosphatidylcholine of 0.1 mM.Approximately 10 μg of purified ICAM-1, or human glycophorin (SigmaChemical Co., St. Louis, Mo.) as a control membrane glycoprotein, wasadded to each ml of dissolved lipids. The protein-lipid-detergentsolution was then dialyzed at 4° C. against 3 changes of 200 volumes of20 mM Tris/0.14 M NaCl, pH 7.2, and one change of HBSS.

Planar membranes were prepared by the method of Brian et al., Proc.Natl. Acad. Sci. 81:6159 (1984). Glass coverslips (11 mm in diameter)were boiled for 15 minutes in a 1:6 dilution of 7× detergent (Linbro),washed overnight in distilled water, soaked in 70% ethanol, and airdried. An 80 μl drop of vesicle suspension containing either ICAM-1 orglycophorin was placed in the bottom of wells in a 24-well clusterplate, and the prepared glass coverslips were gently floated on top.After 20-30 minutes incubation at room temperature, the wells werefilled with HBSS, and the coverslips were inverted to bring the planarmembrane face up. The wells were then washed extensively with HBSS toremove unbound vesicles. The planar membrane surface was never exposedto air.

In the course of experiments with planar membrane fused to glasssurfaces, vesicles containing ICAM-1 were found to bind directly to theplastic surface of multi-well tissue culture plates, and retainfunctional activity as evidenced by specific cell binding. Such vesiclesare hereinafter referred to as “plastic-bound vesicles” (PBV) since thenature of the lipid vesicles bound to the plastic has not beendetermined. Plastic-bound vesicles were prepared by adding 30 μl ofvesicle suspension directly to the bottom of wells in 96-well tissueculture trays (Falcon), followed by incubation and washing as describedfor planar membranes.

Cell Adhesion Assays

Cell adhesion assays using planar membranes or plastic-bound vesicleswere both done in essentially the same way, except that the cell numbersand volumes for PBV assays were reduced to one-fifth that used in planarmembrane assays.

T-lymphocytes from normal controls and a Leukocyte Adhesion Deficiency(LAD) patient whose cells fail to express LFA-1 (Anderson, D. C. et al.,J. Infect. Dis. 152:668 (1985)) were prepared by culturing peripheralblood mononuclear cells with 1 μg/ml Concanavalin-A (Con-A) in RPMI-1640plus 20% FCS at 5×10⁵ cells/ml for 3 days. The cells were then washedtwice with RPMI and once with 5 mM methyl-alpha-D-mannopyranoside toremove residual lectin from the cell surface. The cells were grown inRPMI/20% FCS containing 1 ng/ml recombinant IL-2, and were used between10 and 22 days after the initiation of culture.

To detect cell binding to planar membranes or PBV, Con-A blasts, theT-lymphoma SKW-3, and the EBV-transformed B-lymphoblastoid cell lines JY(LFA-1 positive) and LFA-1 deficient lymphoblastoid cell line (BBN)(derived from patient 1, Springer, T. A. et al., J. Exper. Med.160:1901-1918 (1986) were radiolabeled by incubation of 1×10⁷ cells in 1ml of RPMI-1640/10% FCS with 100 μCi of Na⁵¹CrO₄ for 1 hour at 37° C.,followed by four washes with RPMI-1640 to remove unincorporated label.In monoclonal antibody blocking experiments, cells or plastic-boundvesicles were pretreated with 20 μg/ml of purified antibody inRPMI-1640/10% FCS at 4° C. for 30 minutes, followed by 4 washes toremove unbound antibody. In experiments on the effects of divalentcations on cell binding, the cells were washed once with Ca²⁺, Mg²⁺-freeHBSS plus 10% dialyzed FCS, and CaCl and MgCl were added to theindicated concentrations. In all experiments, cells and planar membranesor PBV were pre-equilibrated at the appropriate temperature (4° C., 22°C., or 37° C.) in the appropriate assay buffer.

To measure cell binding to purified ICAM-1, ⁵¹Cr-labeled cells (5×10⁵EBV-transformants in planar membrane assays; 1×10⁵ EBV-transformants orSKW-3 cells, 2×10⁵ Con-A blasts in PBV assays) were centrifuged for 2minutes at 25×g onto planar membranes or PBV, followed by incubation at4° C., 22° C., or 37° C. for one hour. After incubation, unbound cellswere removed by eight cycles of filling and aspiration with bufferpre-equilibrated to the appropriate temperature. Bound cells werequantitated by solubilization of well contents with 0.1 N NaOH/1% TritonX-100 and counting in a gamma counter. Percent cell binding wasdetermined by dividing cpm from bound cells by input cell-associatedcpm. In planar membrane assays, input cpm were corrected for the ratioof the surface area of coverslips compared to the surface area of theculture wells.

In these assays, EBV-transformed B-lymphoblastoid cells, SKW-3T-lymphoma cells, and Con-A T-lymphoblasts bound specifically to ICAM-1in artificial membranes (FIGS. 11 and 12). The binding was specificsince the cells bound very poorly to control planar membranes orvesicles containing equivalent amounts of another human cell surfaceglycoprotein, glycophorin. Furthermore, LFA-1 positive EBV-transformantsand Con-A blasts bound, while their LFA-1 negative counterparts failedto bind to any significant extent, demonstrating that the binding wasdependent on the presence of LFA-1 on the cells.

Both the specificity of cell binding and the dependence on cellularLFA-1 were confirmed in monoclonal antibody blocking experiments (FIG.13). The binding of JY cells could be inhibited by 97% when theICAM-1-containing PBV were pretreated with anti-ICAM-1 monoclonalantibody RR1/1. Pretreatment of the cells with the same antibody hadlittle effect. Conversely, the anti-LFA-1 monoclonal antibody TS1/18inhibited binding by 96%, but only when the cells, but not the PBV, werepretreated. A control antibody TS2/9 reactive with LFA-3 (a differentlymphocyte surface antigen) had no significant inhibitory effect wheneither cells or PBV were pretreated. This experiment demonstrates thatICAM-1 itself in the artificial membranes, and not some minorcontaminant, mediates the observed cellular adhesion and that theadhesion is dependent on LFA-1 on the binding cell.

The binding of cells to ICAM-1 in artificial membranes also displayedtwo other characteristics of the LFA-1 dependent adhesion system:temperature dependence and a requirement for divalent cations. As shownin FIG. 14, Con-A blasts bound to ICAM-1 in PBV most effectively at 37°C., partially at 22° C., and very poorly at 4° C. As shown in FIG. 15,the binding was completely dependent on the presence of divalentcations. At physiological concentrations, Mg²⁺ alone was sufficient formaximal cell binding, while Ca²⁺ alone produced very low levels ofbinding. However, Mg²⁺ at one-tenth of the normal concentration combinedwith Ca²⁺ was synergistic and produced maximal binding.

In summary, the specificity of cell binding to purified ICAM-1incorporated into artificial membranes, the specific inhibition withmonoclonal antibodies, and the temperature and divalent cationrequirements demonstrate that ICAM-1 is a specific ligand for theLFA-1-dependent adhesion system.

Example 22 Expression of ICAM-1 and HLA-DR in Allergic and Toxic PatchTest Reactions

Skin biopsies of five normal individuals were studied for theirexpression of ICAM-1 and HLA-DR. It was found that while the endothelialcells in some blood vessels usually expressed ICAM-1, there was noICAM-1 expressed on keratinocytes from normal skin. No staining forHLA-DR on any keratinocyte from normal skin biopsies was observed. Thekinetics of expression of ICAM-1 and class II antigens were then studiedon cells in biopsies of allergic and toxic skin lesions. It was foundthat one-half of the six subjects studied had keratinocytes whichexpressed ICAM-1, four hours after application of the hapten (Table 11).There was an increase in the percentage of individuals expressing ICAM-1on their keratinocytes with time of exposure to the hapten as well as anincrease in the intensity of staining indicating more ICAM-1 expressionper keratinocyte up to 48 hours. In fact, at this time point aproportion of keratinocytes in all biopsies stained positively forICAM-1. At 72 hours (24 hours after the hapten was removed), seven ofthe eight subjects still had ICAM-1 expressed on their keratinocyteswhile the expression of ICAM-1 on one subject waned between 48 and 72hours.

TABLE 11 Kinetics of Induction of ICAM-1 and HLA-DR on Keratinocytesfrom Allergic Patch Test Biopsies Time After Patch No. of ICAM-1 HLA-DRICAM-1& Application (h) Biopsies Only Only HLA-DR Normal Skin 5 0 0 0Allergic Patch Test  4 6  3^(a) 0 0  8 9 3 0 0 24 8 7 0 0  48^(b) 8 5 03 72 8 6 0 1 ^(a)Samples were considered as positive if at least smallclusters of keratinocytes were stained. ^(b)All patches were removed atthis time point.

Histologically, the staining pattern for ICAM-1 on keratinocytes frombiopsies taken four hours after application of the hapten was usually insmall clusters. At 48 hours, ICAM-1 was expressed on the surface of themajority of the keratinocytes, no difference being seen between thecenter and periphery of the lesion. The intensity of the stainingdecreased as the keratinocytes approached the stratum corneum. This wasfound in biopsies taken from both the center and the periphery of thelesions. Also at this time point, the patch test was positive(infiltration, erythema and vesicles). No difference in ICAM-1expression was observed when different haptens were applied on sensitiveindividuals. In addition to keratinocytes, ICAM-1 was also expressed onsome mononuclear cells and endothelial cells at the site of the lesion.

The expression of HLA-DR on keratinocytes in the allergic skin lesionswas less frequent than that of ICAM-1. None of the subjects studied hadlesions with keratinocytes that stained positively for HLA-DR up to 24hours after the application of the hapten. In fact, only four biopsysamples had keratinocytes that expressed HLA-DR and no biopsy hadkeratinocytes that was positive for HLA-DR and not ICAM-1 (Table 11).

In contrast to the allergic patch test lesion, the toxic patch testlesion induced with croton oil or sodium lauryl sulfate hadkeratinocytes that displayed little if any ICAM-1 on their surfaces atall time points tested (Table 12). In fact, at 48 hours after the patchapplication, which was the optimum time point for ICAM-1 expression inthe allergic patch test subjects, only one of the 14 toxic patch testsubjects had keratinocytes expressing ICAM-1 in their lesions. Also incontrast to the allergic patch test biopsies, there was no HLA-DRexpressed on keratinocytes of toxic patch test lesions.

These data indicate that ICAM-1 is expressed in immune-basedinflammation and not in toxic based inflammation, and thus theexpression of ICAM-1 may be used to distinguish between immuno based andtoxic based inflammation, such as acute renal failure in kidneytransplant patients where it is difficult to determine whether failureis due to rejection or nephrotoxicity of the immuno-suppressivetherapeutic agent. Renal biopsy and assessment of upregulation of ICAM-1expression will allow differentiation of immune based rejection andnon-immune based toxicity reaction.

TABLE 12 Kinetics of Induction of ICAM-1 and HLA-DR on Keratinocytesfrom Toxic Patch Test Biopsies Time After Patch No. of ICAM-1 HLA-DRICAM-1& Application (h) Biopsies Only Only HLA-DR  4 4 0 0 0  8 3  1^(a)0 0 24 3 1 0 0  48^(b) 14 1 0 0 72 3 1 0 0 ^(a)Samples were consideredas positive if at least small clusters of keratinocytes were stained.^(b)All patches were removed at this time point.

Example 23 Expression of ICAM-1 and HLA-DR in Benign Cutaneous Diseases

Cells from skin biopsies of lesions from patients with various types ofinflammatory skin diseases were studied for their expression of ICAM-1and HLA-DR. A proportion of keratinocytes in biopsies of allergiccontact eczema, pemphigoid/pemphigus and lichen planus expressed ICAM-1.Lichen planus biopsies showed the most intense staining with a patternsimilar to or even stronger than that seen in the 48-hour allergic patchtest biopsies (Table 13). Consistent with results seen in the allergicpatch test, the most intensive ICAM-1 staining was seen at sites ofheavy mononuclear cell infiltration. Furthermore, 8 out of the 11 Lichenplanus biopsies tested were positive for HLA-DR expression onkeratinocytes.

The expression of ICAM-1 on keratinocytes from skin biopsies of patientswith exanthema and urticaria was less pronounced. Only four out of theseven patients tested with these diseases had keratinocytes thatexpressed ICAM-1 at the site of the lesion. HLA-DR expression was onlyfound on one patient and this was in conjunction with ICAM-1.

Endothelial cells and a proportion of the mononuclear cell infiltratefrom all the benign inflammatory skin diseases tested expressed ICAM-1to a varying extent.

TABLE 13 Expression of ICAM-1 and HLA-DR on Keratinocytes from BenignCutaneous Diseases No. of ICAM-1 HLA-DR ICAM-1& Diagnosis Cases OnlyOnly HLA-DR Allergic Contact 5  3^(a) 0 2 Eczema Lichen Planus 11 3 0 8Pemphigoid/ 2 2 0 0 Pemphigus Exanthema 3 2 0 0 Urticaria 4 1 0 1^(a)Samples were considered as positive if at least small clusters ofkeratinocytes were stained.

Example 24 Expression of ICAM-1 on Keratinocytes of Psoriatic SkinLesions

The expression of ICAM-1 in skin biopsies from 5 patients with psoriasiswere studied before the initiation and periodically during a course ofPUVA treatment. Biopsies were obtained from 5 patients with classicalpsoriasis confirmed by histology. Biopsies were taken sequentiallybefore and during indicated time of PUVA treatment. PUVA was given 3 to4 times weekly. Biopsies were taken from the periphery of the psoriaticplaques in five patients and, in addition biopsies were taken fromclinically normal skin in four of the patients.

Fresh skin biopsy specimens were frozen and stored in liquid nitrogen.Six micron cryostat sections were air dried overnight at roomtemperature, fixed in acetone for 10 minutes and either stainedimmediately or wrapped in aluminum foil and stored at −80° C. untilstaining.

Staining was accomplished in the following manner. Sections wereincubated with monoclonal antibodies and stained by a three stageimmunoperoxidase method (Stein, H., et. al., Adv. Cancer Res 42:67-147,(1984)), using a diaminobenzidine H₂O₂, substrate. Tonsils and lymphnodes were used as positive control for anti-ICAM-1 and HLA-DR staining.Tissue stained in the absence of primary antibody were negativecontrols.

The monoclonal antibodies against HLA-DR were purchased from BectonDickinson (Mountainview, Calif.). The monoclonal anti-ICAM-1 antibodywas R6-5-D6. Peroxidase-conjugated rabbit anti-mouse Ig andperoxidase-conjugated swine anti-rabbit Ig were purchased fromDAKAPATTS, Copenhagen, Denmark. Diaminobenzidine-tetrahydrochloride wereobtained from Sigma (St. Louis, Mo.).

The results of the study show that the endothelial cells in some bloodvessels express ICAM-1 in both diseased and normal skin, but theintensity of staining and the number of blood vessels expressing ICAM-1was increased in the psoriatic skin lesions. Moreover, the pattern ofexpression of ICAM-1 in keratinocytes of untreated psoriatic skinlesions from the five patients varied from only small clusters of cellsstaining to many keratinocytes being stained. During the course of PUVAtreatment, the ICAM-1 expression on 2 of the patients (patients 2 and 3)showed marked reduction which preceded or was concurrent with clinicalremission (Table 14). Patients 1, 4 and 5 had decreases and increases ofICAM-1 expression during the PUVA treatment which correlated to clinicalremissions and relapses, respectively. There was no ICAM-1 expression onkeratinocytes from normal skin before or after PUVA treatment. Thisindicates that PUVA does not induce ICAM-1 on keratinocytes from normalskin.

Of note was the observation that the density of the mononuclear cellinfiltrate correlated with the amount of ICAM-1 expression onkeratinocytes. This pertained to both a decreased number of mononuclearcells in lesions during PUVA treatment when ICAM-1 expression also wanedand an increased number of mononuclear cells during PUVA treatment whenICAM-1 expression on keratinocytes was more prominent. Endothelial cellsand dermal mononuclear cells are also ICAM-1-positive. In clinicallynormal skin, ICAM-1 expression was confined to endothelial cells with nolabelling of keratinocytes.

The expression of HLA-DR on keratinocytes was variable. There was noHLA-DR positive biopsy that was not also ICAM-1 positive.

In summary, these results show that before treatment, ICAM-1 expressionis pronounced on the keratinocytes and correlate to a dense mononuclearcellular infiltrate. During PUVA treatment a pronounced decrease of theICAM-1 staining is seen to parallel the clinical improvement.Histologically the dermal infiltrate also diminished. When a clinicalrelapse was obvious during treatment, the expression of ICAM-1 on thekeratinocytes increased, as well as the density of the dermalinfiltrate. When a clinical remission was seen during treatment, therewas a concurrent decrease in ICAM-1 staining on the keratinocytes aswell as decrease in the dermal infiltrate. Thus the expression of ICAM-1on keratinocytes corresponded to the density of the mononuclear cellularinfiltrate of the dermis. These data show that clinical response to PUVAtreatment resulted in a pronounced decrease of ICAM-1 expression onkeratinocytes parallel to a more moderate decline of the mononuclearcells. This indicates that ICAM-1 expression on keratinocytes isresponsible for initiating and maintaining the dermal infiltrate andthat PUVA treatment down regulates ICAM-1 which in turn mitigates thedermal infiltrate and the inflammatory response. The data also indicatesthat there was variable HLA-DR expression on keratinocytes during PUVAtreatment.

The expression of ICAM-1 on keratinocytes of psoriatic lesionscorrelates with the clinical severity of the lesion as well as with thesize of the dermal infiltrate. Thus ICAM-1 plays a central role inpsoriasis and inhibition of its expression and/or inhibition of itsinteraction with the CD 18 complex on mononuclear cells will be aneffective treatment of the disease. Furthermore, monitoring ICAM-1expression on keratinocytes will be an effective tool for diagnosis andprognosis, as well as evaluating the course of therapy of psoriasis.

TABLE 14 Sequential ICAM-1 Expression by Keratinocytes in Psoriatic SkinLesions (PS) and Clinically Normal Skin (N) Before and During PUVATreatment Time before patient no. and during 1 2 3 4 5 PUVA treatment PSPS N PS N PS N PS N 0 + + − ++ − ++ − +++ − 1 day + 1 week + + − − − ++− + − 0 2 weeks ++ + − + − + − 3 weeks ++ * 0 4 weeks ++ + − − − ++− * * 5-6 weeks − − − − 0 7 weeks (++) (+) +++ − * * 10 weeks (+) − −+++ Many positive keratinocytes ++ A proposition of positivekeratinocytes + Focal positive keratinocytes (+) Very few scatteredpositive keratinocytes − No positive staining * Clinical remission 0Clinical relapse

Example 25 Expression of ICAM-1 and HLA-DR in Malignant CutaneousDiseases

Unlike lesions from benign cutaneous conditions, the expression ofICAM-1 on keratinocytes from malignant skin lesions was much morevariable (Table 15). Of the 23 cutaneous T-cell lymphomas studied,ICAM-1 positive keratinocytes were identified in only 14 cases. Therewas a tendency for keratinocytes from biopsies of mycosis fungoideslesions to lose their ICAM-1 expression with progression of the diseaseto more advanced stages. However, ICAM-1 expression was observed on avarying proportion of the mononuclear cell infiltrate from most of thecutaneous T cell lymphoma lesions. Among the remaining lymphomasstudied, four of eight had keratinocytes that expressed ICAM-1. Of the29 patients with malignant cutaneous diseases examined, 5 hadkeratinocytes that expressed HLA-DR without expressing ICAM-1 (Table15).

TABLE 15 Expression of ICAM-1 and HLA-DR on Keratinocytes from MalignantCutaneous Diseases No. of ICAM-1 HLA-DR ICAM-1& Diagnosis Cases OnlyOnly HLA-DR CTCL, MFI 8  1^(a) 0 4 CTCL, MFII-III 10 1 2 5 CTCL, SS 3 10 2 CTCL, Large Cell 2 0 2 0 CBCL 2 0 0 1 Leukemia Cutis 3 1 1 1Histiocytosis X 1 0 0 0 ^(a)Samples were considered as positive if atleast small clusters of keratinocytes were stained.

Example 26 Effect of Anti-ICAM-1 Antibodies on the Proliferation ofHuman Peripheral Blood Mononuclear Cells

Human peripheral blood mononuclear cells are induced to proliferate bythe presence and recognition of antigens or mitogens. Certain molecules,such as the mitogen, concanavalin A, or the T-cell-binding antibodyOKT3, cause a non-specific proliferation of peripheral blood mononuclearcells to occur.

Human peripheral blood mononuclear cells are heterogeneous in that theyare composed of subpopulations of cells which are capable of recognizingspecific antigens. When a peripheral blood mononuclear cell which iscapable of recognizing a particular specific antigen, encounters theantigen, the proliferation of that subpopulation of mononuclear cell isinduced. Tetanus toxoid and keyhole limpet hemocyanin are examples ofantigens which are recognized by subpopulations of peripheralmononuclear cells but are not recognized by all peripheral mononuclearcells in sensitized individuals.

The ability of anti-ICAM-1 monoclonal antibody R6-5-D6 to inhibitproliferative responses of human peripheral blood mononuclear cells insystems known to require cell-cell adhesions was tested.

Peripheral blood mononuclear cells were purified on Ficoll-Paque(Pharmacia) gradients as per the manufacturer's instructions. Followingcollection of the interface, the cells were washed three times with RPMI1640 medium, and cultured in flat-bottomed 96-well microtiter plates ata concentration of 10⁶ cells/ml in RPMI 1640 medium supplemented with10% fetal bovine serum, 2 mM glutamine, and gentamicin (50 μg/ml).

Antigen, either the T-cell mitogen, concanavalin A (0.25 μg/ml); theT-cell-binding antibody, OKT3 (0.001 pg/ml); keyhole limpet hemocyanin(10 g/ml) or tetanus toxoid (1:100 dilution from source) were added tocells which were cultured as described above in either the presence orabsence of anti-ICAM antibody (R6-5-D6; final concentration of 5 g/ml).Cells were cultured for 3.5 days (concanavalin A experiment), 2.5 days(OKT3 experiment), or 5.5 days (keyhole limpet hemocyanin and tetanustoxoid experiments) before the assays were terminated.

Eighteen hours prior to the termination of the assay, 2.5 μCi of³H-thymidine was added to the cultures. Cellular proliferation wasassayed by measuring the incorporation of thymidine into DNA by theperipheral blood mononuclear cells. Incorporated thymidine was collectedand counted in a liquid scintillation counter (Merluzzi et al., J.Immunol. 139:166-168 (1987)). The results of these experiments are shownin FIG. 16 (concanavalin A experiment), FIG. 17 (OKT3 experiment), FIG.18 (keyhole limpet hemocyanin experiment), and FIG. 19 (tetanus toxoidexperiment).

It was found that anti-ICAM-1 antibody inhibits proliferative responsesto the non-specific T-cell mitogen, ConA; the non-specific T-cellassociated antigen, OKT-3; and the specific antigens, keyhole limpethemocyanin and tetanus toxoid, in mononuclear cells. The inhibition byanti-ICAM-1 antibody was comparable to that of anti-LFA-1 antibodysuggesting that ICAM-1 is a functional ligand of LFA-1 and thatantagonism of ICAM-1 will inhibit specific defense system responses.

Example 27 Effect of Anti-ICAM-1 Antibody on the Mixed LymphocyteReaction

As discussed above, ICAM-1 is necessary for effective cellularinteractions during an immune response mediated through LFA-1-dependentcell adhesion. The induction of ICAM-1 during immune responses orinflammatory disease allows for the interaction of leukocytes with eachother and with endothelial cells.

When lymphocytes from two unrelated individuals are cultured in eachothers presence, blast transformation and cell proliferation of thelymphocytes are observed. This response, of one population oflymphocytes to the presence of a second population of lymphocytes, isknown as a mixed lymphocyte reaction (MLR), and is analogous to theresponse of lymphocytes to the addition of mitogens (Immunology TheScience of Self-Nonself Discrimination, Klein, J., John Wiley & Sons, NY(1982), pp 453-458).

Experiments were performed to determine the effect of anti-ICAMmonoclonal antibodies on the human MLR. These experiments were conductedas follows. Peripheral blood was obtained from normal, healthy donors byvenipuncture. The blood was collected in heparinized tubes and diluted1:1 at room temperature with Puck's G (GIBCO) balanced salt solution(BSS). The blood mixture (20 ml) was layered over 15 ml of aFicoll/Hypaque density gradient (Pharmacia, density=1.078, roomtemperature) and centrifuged at 1000×g for 20 minutes. The interface wasthen collected and washed 3× in Puck's G. The cells were counted on ahemacytometer and resuspended in RPMI-1640 culture medium (GIBCO)containing 0.5% of gentamicin, 1 mM L-glutamine (GIBCO) and 5% heatinactivated (56° C., 30 min.) human AB sera (Flow Laboratories)(hereafter referred to as RPMI-culture medium).

Mouse anti-ICAM-1 (R6-5-D6) was used in these experiments. Allmonoclonal antibodies (prepared from ascites by Jackson ImmunoResearchLaboratories, Boston, Mass.) were used as purified IgG preparations.

Peripheral blood mononuclear cells (PBMC) were cultured in medium at6.25×10⁵ cells/ml in Linbro round-bottomed microliter plates(#76-013-05). Stimulator cells from a separate donor were irradiated at1000 R and cultured with the responder cells at the same concentration.The total volume per culture was 0.2 ml. Controls included respondercells alone as well as stimulator cells alone. The culture plates wereincubated at 37° C. in a 5% CO₂-humidified air atmosphere for 5 days.The wells were pulsed with 0.5 μCi of tritiated thymidine (3HT) (NewEngland Nuclear) for the last 18 hours of culture. In some cases atwo-way MLR was performed. The protocol was the same except that thesecond donor's cells were not inactivated by irradiation.

The cells were harvested onto glass fiber filters using an automatedmultiple sample harvester (Skatron, Norway), rinsing with water andmethanol. The filters were oven dried and counted in Aquasol in aBeckman (LS-3801) liquid scintillation counter. Results are reported asthe Mean CPM t standard error of 6 individual cultures.

Table 16 shows that purified anti-ICAM-1 monoclonal antibodies inhibitedthe MLR in a dose dependent manner with significant suppression apparentat 20 ng/ml. Purified mouse IgG had little or no suppressive effect.Suppression of the MLR by the anti-ICAM-1 monoclonal antibody occurswhen the antibody is added within the first 24 hours of cultures (Table16).

TABLE 16 Effect of Anti-ICAM-1 Antibody on the One-Way LymphocyteReaction Responder Stimulator Cells^(a) Cells^(b) Antibody^(c) ³HTIncorporation (CPM) − − —  445^(d) ± 143 − + —   148 ± 17 + − —   698 ±72 + + — 42,626 ± 1,579 + + mIgG (10.0 μg) 36,882 ± 1,823 (14%) + + mIgG(0.4 μg) 35,500 ± 1,383 (17%) + + mIgG (0.02 μg) 42,815 ± 1,246 (0%) + +R6-5-D6 (10.0 μg)  8,250 ± 520 (81%) + + R6-5-D6 (0.4 μg) 16,142 ± 858(62%) + + R6-5-D6 (0.03 μg) 28,844 ± 1,780 (32%) ^(a)Responder cells(6.25 × 10⁵/ml) ^(b)Stimulator Cells (6.25 × 10⁵/ml, irradiated at1000R) ^(c)Purified Monoclonal Antibody to ICAM-1 (R6-5-D6) or purifiedmouse IgG (mIgG) at final concentrations (ug/ml). ^(d)Mean ± S.E. of 5-6cultures, numbers in parentheses indicate percent inhibition of MLR

TABLE 17 Time of Addition of Anti-ICAM-1 ³HT Incorporation (CPM) Time ofAddition of Medium or Antibody R^(a) S^(b) Additions^(C) Day 0 Day 1 Day2 − − medium 205^(d) ± 14   476 ± 132  247 ± 75 − + medium  189 ± 16nd^(e) nd + − medium 1,860 ± 615  nd nd + + medium 41,063 ± 2,940 45,955± 2,947 50,943 ± 3,072 + + R6-5-D6 17,781 ± 1,293 38,409 ± 1,681 47,308± 2,089 (57%)^(f) (16%) (7%) ^(a)Responder cells (6.25 × 10⁵/ml)^(b)Stimulator Cells (6.25 × 10⁵/ml, irradiated at 1000R) ^(c)CultureMedium or Purified Monoclonal Antibody to ICAM-1 (R6-5-D6) at 10 μg/mlwere added on day 0 at 24 hour intervals ^(d)Mean ± S.E. of 4-6 cultures^(e)nd = not done ^(f)Percent Inhibition

In summary, the ability of antibody against ICAM-1 to inhibit the MLRshows that ICAM-1 monoclonal antibodies have therapeutic utility inacute graft rejection. ICAM-1 monoclonal antibodies also havetherapeutic utility in related immune mediated disorders dependent onLFA-1/ICAM-1 regulated cell to cell interactions.

The experiments described here show that the addition of monoclonalantibodies to ICAM-1 inhibit the mixed lymphocyte reaction (MLR) whenadded during the first 24 hours of the reaction. Furthermore, ICAM-1becomes upregulated on human peripheral blood monocytes during in vitroculture.

Furthermore, it was found that ICAM-1 is not expressed on resting humanperipheral blood lymphocytes or monocytes. ICAM-1 is up regulated on themonocytes of cultured cells alone or cells co-cultured with unrelateddonor cells in a mixed lymphocyte reaction using conventional flowcytometric analyses. This up regulation of ICAM-1 on monocytes can beused as an indicator of inflammation, particularly if ICAM-1 isexpressed on fresh monocytes of individuals with acute or chronicinflammation.

ICAM-1's specificity for activated monocytes and the ability of antibodyagainst ICAM-1 to inhibit an MLR suggest that ICAM-1 monoclonalantibodies may have diagnostic and therapeutic potential in acute graftrejection and related immune mediated disorders requiring cell to cellinteractions.

Example 28 Synergistic Effects of the Combined Administration ofAnti-ICAM-1 and Anti-LFA-1 Antibodies

As shown in Example 27, the MLR is inhibited by anti-ICAM-1 antibody.The MLR can also be inhibited by the anti-LFA-1 antibody. In order todetermine whether the combined administration of anti-ICAM-1 andanti-LFA-1 antibodies would have an enhanced, or synergistic effect, anMLR assay (performed as described in Example 27) was conducted in thepresence of various concentrations of the two antibodies.

This MLR assay revealed that the combination of anti-ICAM-1 andanti-LFA-1, at concentrations where neither antibody alone dramaticallyinhibits the MLR, is significantly more potent in inhibiting the MLRresponse (Table 18). This result indicates that therapies whichadditionally involve the administration of anti-ICAM-1 antibody (orfragments thereof) and anti-LFA-1 antibody (of fragments thereof) havethe capacity to provide an improved anti-inflammatory therapy. Such animproved therapy permits the administration of lower doses of antibodythan would otherwise be therapeutically effective, and has importance incircumstances where high concentrations of individual antibodies inducean anti-idiotypic response.

TABLE 18 Effect of Various Doses of Anti-ICAM-1 and (R3.1) Anti-LFA-1 onMixed Lymphocyte Reaction Concentration % Inhibition (ug/ml) Anti-ICAM-1(R6-5-D6) Anti-LFA-1 0 .004 .02 .1 .5 2.5 0.0 0 7 31 54 69 70 0.0008 1 728 48 62 71 0.004 0 13 30 50 64 72 0.02 29 38 64 75 84 86 0.1 92.5 90 9192 92 92 0.5 93 90 90 92 93 91

Example 29 Additive Effects of Combined Administration of Sub-optimalDoses Anti-ICAM-1 and Other Immunosuppressive Agents in the MLR

As shown in Example 28, the MLR is inhibited by combinations ofanti-ICAM-1 and anti-LFA-1 antibodies. In order to determine whether thecombined administration of anti-ICAM-1 and other immunosuppressiveagents (such as dexamethasone, azathioprine, cyclosporin A or steroids(such as, for example, prednisone, etc.) would also have enhancedeffects, MLR assays were performed using sub-optimal concentrations(i.e. concentrations which would be lower than the optimal concentrationat which the agent alone would be provided to a subject) of R6-5-D6 inconjunction with other immunosuppressive agents as per the protocol inExample 27.

The data indicate that the inhibitory effects of R6-5-D6 are at leastadditive with the inhibitory effects of suboptimal doses ofdexamethasone (Table 19), Azathioprine (Table 20) and cyclosporin A(Table 21). This implies that anti-ICAM-1 antibodies can be effective inlowering the necessary doses of known immunosuppressants, thus reducingtheir toxic side effects. In using an anti-ICAM-1 antibody (or afragment thereof) to achieve such immunosuppression, it is possible tocombine the administration of the antibody (or fragment thereof) witheither a single additional immunosuppressive agent, or with acombination of more than one additional immunosuppressive agent.

TABLE 19 Effect of Anti-ICAM-1 and Dexamethasone on the Human MLR ³HTInhibitor Incorporation % Group (ng/ml) (CPM) Inhibition Media — 156 —Stimulators (S) — 101 — Responders (R) — 4,461 — R × S — 34,199 — R × SR6-5-D6 (8) 26,224 23 R × S Dex (50) 14,158 59 R × S R6-5-D6 (8) + Dex(50) 7,759 77 Dex: Dexamethasone

TABLE 20 Effect of Anti-ICAM-1 and Azathioprine on the Human MLR ³HTInhibitor Incorporation % Group (ng/ml) (CPM) Inhibition Media — 78 —Stimulators (S) — 174 — Responders (R) — 3,419 — R × S — 49,570 — R × SR6-5-D6 (8) 44,374 11 R × S Azathioprine (1) 42,710 14 R × S R6-5-D6(8) + 34,246 31 Azathioprine (1)

TABLE 21 Effect of Anti-ICAM-1 and Cyclosporin A on the Human MLR ³HTInhibitor Incorporation % Group (ng/ml) (CPM) Inhibition Media — 87 —Stimulators (S) — 206 — Responders (R) — 987 — R × S — 31,640 — R × SR6-5-D6 (8) 26,282 17 R × S CyA (10) 23,617 25 R × S R6-5-D6 (8) + CyA(10) 19,204 39 CyA: Cyclosporin A

Example 30 Effect of Anti-ICAM-1 Antibody in Suppressing the Rejectionof Transplanted Allogeneic Organs

In order to demonstrate the effect of anti-ICAM-1 antibody insuppressing the rejection of an allogeneic transplanted organ,Cynomolgus monkeys were transplanted with allogeneic kidneys accordingto the method described by Cosimi et al. (Transplant. Proc. 13:499-503(1981)) with the modification that valium and ketamine were used asanesthesia.

Thus, the kidney transplantation was performed essentially as follows.Heterotropic renal allografts were performed in 3-5 kg Cynomolgusmonkeys, essentially as described by Marquet (Marquet et al., MedicalPrimatology, Part II, Basel, Karger, p. 125 (1972)) after induction ofanesthesia with valium and ketamine. End-to-side anastomoses of donorrenal vessels on a patch of aorta or vena cava were constructed using7-0 Prolene suture. The donor ureter was spatulated and implanted intothe bladder by the extravesical approach (Taguchi, Y., et al., inDausset et al. (eds.), in: Advances in Transplantation, Baltimore,Williams & Wilkins, p. 393 (1968)). Renal function was evaluated byweekly or biweekly serum creatinine determinations. In addition,frequent allograft biopsies were obtained for histopathologicexamination and complete autopsies were performed on all nonsurvivingrecipients. In most recipients, bilateral nephrectomy was performed atthe time of transplantation and subsequent uremic death was consideredthe end point of allograft survival. In some recipients, unilateralnative nephrectomy and contralateral ureteral ligation were performed atthe time of transplantation. When allograft rejection occurred, theligature on the autologous ureter was then removed resulting inrestoration of normal renal function and the opportunity to continueimmunologic monitoring of the recipient animal.

Monoclonal antibody R6-5-D6 was administered daily for 12 days startingtwo days prior to transplant at a dose of 1-2 mg/kg/day. Serum levels ofcreatinine were periodically tested to monitor rejection. The effect ofanti-ICAM-1 antibody on the immune system's rejection of the allogeneickidneys is shown in Table 22.

TABLE 22 R6-5-D6 Activity in Prolonging Renal Allograft Survival inProphylactic Protocols in the Cynomolgus Monkey^(a) Days of Survival/Monkey Dose of R6-5-D6 (mg/kg) Post-Treatment Control 1 —  8 Control 2 —11 Control 3 — 11 Control 4 — 10 Control 5 —  9 Control 6 — 10 M15 1.020 M19 1.0   7^(b) M17 1.0 30 M25 1.5 29 M23 1.0  11^(c) M27 2.0 34 M70.5 22 M11 0.5 26 M10 0.5 22 M8 0.5  26^(d) ^(a)Monkeys were givenR6-5-D6 for 12 consecutive days starting at 2 days prior totransplantation. ^(b)Cause of death is unknown. There was evidence oflatent malaria. ^(c)Died of kidney infarct. ^(d)Still living as of Aug.15, 1988.

The results show that R6-5-D6 was effective in prolonging the lives ofmonkeys receiving allogenic kidney transplants.

Example 31 Effect of Anti-ICAM-1 Antibody in Suppressing Acute Rejectionof Transplanted Organs

In order to show that anti-ICAM-1 antibody is effective in an acutemodel of transplant rejection, R6-5-D6 was also tested in a therapeuticor acute kidney rejection model. In this model, monkey kidneys weretransplanted (using the protocol described in Example 30) and givenpreoperatively 15 mg/kg cyclosporin A (CyA) i.m. until stable renalfunction was achieved. The dose of CyA was then reduced biweekly in 2.5mg/kg increments until rejection occurred as indicated by a rise inblood creatinine levels. At this point, R6-5-D6 was administered for 10days and the length of survival was monitored. It is important to notethat in this protocol, the dose of CyA remains suboptimal since it doesnot change once the acute rejection episode occurs. In this modelhistorical controls (N=5) with no antibody rescue survive 5-14 days fromthe onset of the rejection episode. To date, six animals were testedusing R6-5-D6 in this protocol (Table 23). Two of these animals arestill surviving (M12, 31 days and M5, 47 days following theadministration of R6-5-D6). Two animals lived 38 and 55 days followinginitiations of R6-5-D6 therapy and two animals died from causes otherthan acute rejection (one animal died of CyA toxicity and the other diedwhile being given R6-5-D6 under anesthesia). This model more closelyapproximates the clinical situation in which R6-5-D6 would be initiallyused.

TABLE 23 R6-5-D6 Activity in Prolonging Renal Allograft Survival inTherapeutic Protocols in the Cynomolgus Monkey^(a) Days of Survival/Monkey Day of Rejection Episode^(b) Post-Treatment Controls^(c) 14-985-14 M24 41 38 M21 34   4^(d) M3 41 55 M9 12  11^(e) M12 37 >31^(f ) M526 >47^(f ) ^(a)Monkeys were given 1-2 mg/kg of R6-5-D6 for 10consecutive days following onset of rejection. ^(b)Day at whichcreatinine levels increased as a result of reduction of CyA dosage andR6-5-D6 therapy started. ^(c)Five animals were tested using thetherapeutic protocol described above except that there was no rescuetherapy. Days of survival/post treatment represents days of survivalonce creatinine levels started to rise. ^(d)Died while under anesthesia.Creatinine levels were low. ^(e)Died of CyA toxicity. Creatinine levelswere low. ^(f)Still living as of Aug. 15, 1988.

Example 32 Genetic Construction and Expression of Truncated Derivativesof ICAM-1

In its natural state, ICAM-1 is a cell membrane-bound protein containingan extracellular region of 5 immunoglobulin-like domains, atransmembrane domain, and a cytoplasmic domain. It was desirable toconstruct functional derivatives of ICAM-1 lacking the transmembranedomain and/or the cytoplasmic domain in that a soluble, secreted form ofICAM-1 could be generated. These functional derivatives were constructedby oligonucleotide-directed mutagenesis of the ICAM-1 gene, followed byexpression in monkey cells after transfection with the mutant gene.

Mutations in the ICAM-1 gene resulting in amino acid substitutionsand/or truncated derivatives were generated by the method of Kunkel, T.,(Proc. Natl. Acad. Sci. (U.S.A.) 82:488-492 (1985)). ICAM-1 cDNAprepared as described above was digested with restriction endonucleasesSal 1 and Kpn 1, and the resulting 1.8 kb DNA fragment was subclonedinto the plasmid vector CDM8 (Seed, B. et al., Proc. Natl. Acad. Sci.(U.S.A.) 84:3365-3369 (1987)). A dut⁻, unq⁻ strain of E. coli (BW313/P3)was then transformed with this construct, designated pCD1.8C. Asingle-strand uracil-containing template was rescued from thetransformants by infection with the helper phage R408 (Stratagene®).Mutant ICAM-1 cDNAs were then generated by priming a second strandsynthesis with an oligonucleotide possessing mismatched bases, andsubsequent transformation of a unq⁺ host (MC1061/P3) with the resultingheteroduplex. Mutants were isolated by screening for newly createdendonuclease restriction sites introduced by the mutant oligonucleotide.The mutant ICAM-1 protein was expressed by transfection of Cos-7 cellswith the mutant DNA in the eukaryotic expression vector CDM8 usingstandard DEAE-Dextran procedures (Selden, R. F. et al., In: CurrentProtocols in Molecular Biology (Ausubel, F. M. et al., eds.) pages9.2.1-9.2.6 (1987)).

A truncated functional derivative of ICAM-1 lacking the transmembraneand cytoplasmic domains, but containing the extracellular regionpossessing all 5 immunoglobulin-like domains was prepared. A 30 bpmutant oligonucleotide (CTC TCC CCC CGG TTC TAG ATT GTC ATC ATC) wasused to transform the codons for amino acids tyrosine (Y) and glutamicacid (E) at positions 452 and 453, respectively, to a phenylalanine (F)and a translational stop codon (TAG). The mutant was isolated by itsunique Xba 1 restriction site, and was designated Y⁴⁵²E/F, TAG.

To express the mutant protein, COS cells were transfected with threemutuant subclones (#2, #7, and #8). Three days after transfection withthe three mutant subclones, culture supernates and cell lysate wereanalysed by immunoprecipitation with anti-ICAM-1 monoclonal antibodyRR1/1 and SDS-PAGE. ICAM-1 was precipitated from the culture supernatesof cells transfected with mutant subclones #2 and #8, but not fromdetergent lysates of those cells. The molecular weight of ICAM-1 foundin the culture supernate was reduced approximately 6 kd relative to themembrane form of ICAM-1, which is consistent with the size predictedfrom the mutant DNA. Thus, this functional derivative of ICAM-1 isexcreted as a soluble protein. In contrast, ICAM-1 was notimmunoprecipitated from control culture supernates of cells transfectedwith native ICAM-1, demonstrating that the membrane form of ICAM-1 isnot shed from Cos cells. Furthermore, no ICAM-1 was immunoprecipitatedfrom either culture supernates or cell lysates from negative controlmock-transfected cells.

The truncated ICAM-1 secreted from transfected cells was purified byimmunoaffinity chromatography with an ICAM-1 specific antibody (R6-5-D6)and tested for functional activity in a cell binding assay. Afterpurification in the presence of the detergent octylglucoside,preparations containing native ICAM-1 or the truncated, secreted formwere diluted to a final concentration of 0.25% octylglucoside (aconcentration below the critical micelle concentration of thedetergent). These preparations of ICAM-1 were allowed to bind to thesurfaces of plastic 96-well plates (Nunc), to produce ICAM-1 bound to asolid-phase. After washing out unbound material, approximately 75-80%and 83-88% of SKW-3 cells bearing LFA-1 on their surface boundspecifically to the native and to the truncated forms of ICAM-1,respectively. These data demonstrate that the secreted, truncatedsoluble ICAM-1 functional derivative retained both the immunologicalreactivity and the ability to mediate ICAM-1 dependent adhesion whichare characteristic of native ICAM-1.

A functional derivative of ICAM-1 lacking only the cytoplasmic domainwas prepared by similar methods. A 25 bp oligonucleotide (TC AGC ACG TACCTC TAG MAC CGC CA) was used to alter the codon for amino acid 476 (Y)to a TAG translational stop codon. The mutant was designated Y⁴⁷⁶/TAG.Immunoprecipitation analysis and SDS-PAGE of Cos cells transfected withthe mutant detected a membrane bound form of ICAM-1 with a molecularweight approximately 3 kd less than native ICAM-1. Indirectimmunofluorescence of the mutant-transfected Cos cells demonstrated apunctuate staining pattern similar to naive ICAM-1 expressed onLPS-stimulated human endothelial cells. Finally, cells transfected withthe mutant DNA specifically bound to purified LFA-1 on plastic surfacesin a manner similar to Cos cells transfected with native ICAM-1 DNA(Table 24).

TABLE 24 Ability of Cells Expressing ICAM-1 or a Functional Derivativeof ICAM-1 to Bind LFA-1 % of Cells Expressing ICAM-1 that Bind LFA-1 inthe Presence of: TRANSFECTION No Antibody RR1/1 Mock 0 0 Native ICAM-120 0 Y⁴⁷⁶/TAG 20 0

Example 33 Mapping of ICAM-1 Functional Domains

Studies of ICAM-1 have revealed that the molecule possesses 7 domains.Five of these domains are extracellular (domain 5 being closest to thecell surface, domain 1 being furthest from the cell surface), one domainis a transmembrane domain, and one domain is cytoplasmic (i.e. lieswithin the cell). In order to determine which domains contribute to theability of ICAM-1 to bind LFA-1, epitope mapping studies may be used. Toconduct such studies, different deletion mutants are prepared andcharacterized for their capacity to bind to LFA-1. Alternatively, thestudies may be accomplished using anti-ICAM antibody known to interferewith the capacity of ICAM-1 to bind LFA-1. Examples of such suitableantibody include RR1/1 (Rothlein, R. et al., J. Immunol. 137:1270-1274(1986)), R6.5 (Springer, T. A. et al., U.S. patent application Ser. No.07/250,446), LB-2 (Clark, E. A. et al., In: Leukocyte Typing I (A.Bernard, et al., Eds.), Springer-Verlag pp 339-346 (1984)), or CL203(Staunton, D. E. et al., Cell 56:849-853 (1989)).

Deletion mutants of ICAM-1 can be created by any of a variety of means.It is, however, preferable to produce such mutants via site directedmutagenesis, or by other recombinant means (such as by constructingICAM-1 expressing gene sequences in which sequences that encodeparticular protein regions have been deleted. Procedures which may beadapted to produce such mutants are well known in the art. Using suchprocedures, three ICAM-1 deletion mutants were prepared. The firstmutant lacks amino acid residues F185 through P284 (i.e. deletion ofdomain 3). The second mutant lacks amino acid residues P284 through R451(i.e. deletion of domains 4 and 5). The third mutant lacks amino acidresidues after Y476 (i.e. deletion of cytoplasmic domain). The resultsof such studies indicate that domains 1, 2, and 3 are predominantlyinvolved in ICAM-1 interactions with anti-ICAM-1 antibody and LFA-1.

Example 34 Effect Of Mutations in ICAM-10N LFA-1 Binding

The ability of ICAM-1 to interact with and bind to LFA-1 is mediated byICAM-1 amino acid residues which are present in domains 1 of the ICAM-1molecule (FIGS. 8, 9 and 10). Such interactions are assisted, however,by contributions from amino acids present in domains 2 and 3 of ICAM-1.Thus, among the preferred functional derivatives of the presentinvention are soluble fragments of the ICAM-1 molecule which containdomains 1, 2, and 3 of ICAM-1. More preferred are soluble fragments ofthe ICAM-1 molecule which contain domains 1 and 2 of ICAM-1. Mostpreferred are soluble fragments of the ICAM-1 molecule which containdomain 1 of ICAM-1. Several amino acid residues within the first ICAM-1domain are involved in the interaction of ICAM-1 and LFA-1.Substitutions of these amino acids with other amino acids alter theability of ICAM-1 to bind LFA-1. These amino acid residues and thesubstitutions are shown in Table 25. Table 25 shows the effects of suchmutations on the ability of the resulting mutant ICAM-1 molecule to bindto LFA-1. In Table 25, residues are described with reference to the oneletter code for amino acids, followed by the position of the residue inthe ICAM-1 molecule. Thus, for example, “E90” refers to the glutamicacid residue at position 90 of ICAM-1. Similarly, “E90V” refers to thedipeptide composed of the glutamic acid residue at position 90 and thevaline residue at position 91. The substitution sequence is indicated tothe right of the slash (“/”) mark. The S3VS, V4, R13, D26QPK, Q27, E34,G46NN, K50V, Q58EDS, D60, D71GQS, Q73, Y83, E90, N103, A115N, andN175TSA residues of ICAM-1, for example, are involved in LFA-1 binding(Table 25).

Replacement of these amino acids altered the capacity of ICAM-1 to bindto LFA-1. For example, replacement of V4 with G results in the formationof a mutant ICAM-1 molecule which is less able to bind to LFA-1 (Table25). Replacement of the R13 residue of ICAM-1 with E, or of the E34residue with A, leads to the formation of a mutant molecule withsubstantially less capacity to bind LFA-1. (Table 25). Replacement ofthe D60S residues of ICAM-1 with KL leads to the formation of a mutantmolecule having substantially less capacity to bind LFA-1 (Table 25). Incontrast, replacement of the Q58 residue of ICAM-1 with H leads to theformation of a mutant molecule having a substantially normal capacity tobind LFA-1 (Table 25).

Glycosylation sites in the second domain are also involved in LFA-1binding (Table 25). Replacement of N103 with K, or A115N with SV,results in the formation of a mutant ICAM-1 molecule which issubstantially incapable of binding LFA-1. In contrast, replacement ofthe glycosylation site N175 with A did not appear to substantiallyeffect the capacity of the mutant ICAM-1 to bind LFA-1.

Mutations in the third ICAM-1 domain did not appreciably alterICAM-1-LFA-1 binding (Table 25).

Example 35 Multimeric Forms of ICAM-1 with Increased BiologicalHalf-Life, Affinity and Clearance Ability

Chimeric molecules are constructed in which domains 1 and 2 of ICAM-1are attached to the hinge region of the immunoglobulin heavy chain.Preferred constructs attach the C-terminus of ICAM-1 domain 2 to asegment of the immunoglobulin heavy chain gene just N-terminal to thehinge region, allowing the segmental flexibility conferred by the hingeregion. The ICAM-1 domains 1 and 2 will thus replace the Fab fragment ofan antibody. Attachment to heavy chains of the IgG class and expressionin animal cells will result in the production of a chimeric molecule.Production of molecules containing heavy chains derived from IgA or IgMwill result in expression of molecules containing from 2 to 12 ICAM-1molecules. A chimeric immunoglobulin having an IgG heavy chain willcontain 1-2 ICAM-1 molecules. Co-expression of J-chain gene in theanimal cells producing the ICAM-1 heavy chain chimeric molecules willallow proper assembly of IgA and IgM multimers resulting predominantlyin IgA molecules containing 4 to 6 ICAM-1 molecules and in the case ofIgM containing approximately 10 ICAM-1 molecules.

These chimeric molecules may have several advantages. First, Igmolecules are designed to be long lasting in the circulation and thismay improve biological half-life. Furthermore, the multimeric nature ofthese engineered molecules will allow them to interact with higheravidity with rhinovirus as well as with cell surface LFA-1, depending onthe therapeutic context, and thus greatly decrease the amount ofrecombinant protein which needs to be administered to give an effectivedose. IgA and IgM are highly glycosylated molecules normally present insecretions in mucosal sites as in the nose. Their highly hydrophilicnature helps to keep bacteria and viruses to which they bind out in themucosa, preventing attachment to cells and preventing crossing of theepithelial cell membrane barrier. Thus, they may have increasedtherapeutic efficacy. IgM and in particularly IgA are stable in mucosalenvironments and they may increase the stability of the ICAM-1constructs. If such an ICAM-1 functional derivative is administered inthe blood stream, it may also increase biological half-life. IgA doesnot fix complement and thus would be ideal for applications in whichthis would be deleterious. If IgG H chain chimerics are desired, itwould be possible to mutate regions involved in attachment to complementas well as in interactions with Fc receptors.

Example 36 ICAM-1 Outline Structure and the LFA-1 and Rhinovirus BindingSites Viral Mimicry of a Cell Adhesion Receptor

ICAM-1 and a second LFA-1 counter-receptor, ICAM-2, constitute asubfamily of the immunoglobulin (Ig) superfamily (Staunton, D. E., etal., Cell 52:925-933 (1988), which reference is incorporated herein byreference). ICAM-1 possesses five Ig-like C domains whereas ICAM-2possesses two, which are most homologous to the amino terminal domainsof ICAM-1. ICAM-1 and ICAM-2, expressed on a variety of cell types,support various leukocyte adhesion dependent functions includinginduction and effector functions in the immune response. ICAM-1expression is highly inducible by cytokines and thus the LFA-1/ICAM-1adhesion system is able to guide leukocyte migration and localizationduring inflammation (Rothlein, R. J. Immunol. 137:1270-1274 (1986);Marlin, S. D. et al., Cell 51:813-819 (1987); Kishimoto, T. K. et al.,Adv. Immunol. 46:149-182 (1989); Dustin, M. L. et al., Immunol. Today9:213-215 (1988), all of which references are incorporated herein byreference).

LFA-1 (CD11a/CD18) is a member of the integrin family most closelyrelated to two other leukocyte integrins Mac-1 (CR3; CD11b/CD18) andp150/95 (CD11c/CD18) (Hynes, R. O., Cell 48:549-554 (1987)) Mac-1, inaddition to supporting neutrophil adhesion, has been demonstrated tobind several ligands including iC3b, leishmania gp63 and fibrinogen(Ruoslahti, E., et al., Cell 44:517-518 (1986); Hynes, R. O., Cell48:549-554 (1987)). Binding to these ligands can be competed withpeptides containing either an RGD or KXXDS sequence (Marlin, S. D. etal., Cell 51:813-819 (1987)). Neither ICAM possesses an RGD or KXXDSsequence. It is therefore consistent that interaction between ICAM-1 andLFA-1 is not competed with RGD peptides. Thus, the site of contact onICAM-1 with LFA-1 is not apparent by analogy to many otherintegrin-ligand interactions.

ICAM-1 has recently been shown to be subverted as a receptor by themajor group of rhinoviruses (Greve, J. M. et al., Cell 56:839-847(1989); Staunton, D. E. et al., Cell 56:849-853 (1989); Tomassini, J. E.et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:4907-4911 (1989), whichreferences are incorporated herein by reference). Rhinoviruses, membersof the small, RNA-containing, protein-encapsidated picornavirus family,cause 40-50% of common colds (Rueckert, R. R., In: Fields Virology,Fields, B. N. et al. (eds.), Raven Press, NY, (1985) pp 705-738;Sperber, S. J. et al. Antimicr. Agents Chemo. 32: 409-419 (1988), whichreferences are incorporated herein by reference). Over 100immunologically non-crossreactive rhinoviruses have been defined, ofwhich 90% bind to ICAM-1.

X-ray crystallography shows that rhinoviruses are 30 nm in diameter andhave icosohedral symmetry with 60 copies of each capsid protein(Rossmann, M. G. et al., Nature 317:145-153 (1985)) and hence have 60potential binding sites for ICAM-1. Based on amino acid substitutionmutants, and conformational changes induced by the binding of anti-viraldrugs, a deep region of depression or canyon in the capsid which runsabout its 5-fold axes has been identified (Rossmann, M. G. et al.,Nature 317:145-153 (1985); Colonno, R. J. et al., Proc. Natl. Acad. Sci.(U.S.A.) 85:5449-5453 (1988); Rossmann, M. G. et al., Ann. Rev. Biochem.58:533-573 (1989)). Residues at the floor of the canyon are implicatedin ICAM-1 binding function.

A single ICAM-11 g-like domain is predicted to be of approximately thecorrect dimensions to associate with HRV residues at the canyon floor(Staunton, D. E. et al., Cell 56:849-853 (1989)); however, an antibodyFab fragment is predicted to be excluded (Rossmann, M. G. et al., Nature317:145-153 (1985)). Because the antibody combining site of an Fabfragment is too large to come in contact with the canyon floor, receptorspecificity may be maintained by relatively conserved residues at thecanyon floor while mutations of residues at the canyon rim may allow fornew serotypes and evasion of immune surveillance; the “canyonhypothesis” (Rossmann, M. G. et al., Nature 317:145-153 (1985); Colonno,R. J. et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:5449-5453 (1988);Rossmann, M. G. et al., Ann. Rev. Biochem. 58:533-573 (1989)).

The overall size and shape of ICAM-1 is important to understanding howits Ig domains are arranged. Thus far X-ray crystal structures for Igsuperfamily members are available only for immunoglobulins and HLAantigens, which have paired Ig domains; however, domains can also beunpaired as evidenced by Thy-1 which contains a single domain.

Three non-cross blocking ICAM-1 MAbs (RR1/1, R6.5, and LB-2) which blockbinding to LFA-1 also block HRV binding whereas another (CL203) blocksneither LFA-1 nor HRV binding (Makgoba, M. W., et al., In: Immunobiologyof HLA Volume II: Immunogenetics and HistocomDatibility, B. Dupont, ed.,New York: Springer-Verlag, pp. 577-580 (1989); Maio, M., J. Immunol.143:181-188 (1989); Staunton, D. E. et al., Cell 56:849-853 (1989),which references are incorporated herein by reference). This findingshows that LFA-1 and HRV may bind to an overlapping region on ICAM-1.

Besides ICAM-1, the cell adhesion molecule CD4 and the complementreceptor CR2 have recently been found to be subverted as virus receptorsby HIV and EBV viruses, respectively (Maddon, P. J., Cell 47:333-348(1986); Fingeroth, J. D., et al., Proc. Natl. Acad. Sci. USA81:4510-4514 (1984), which references are incorporated herein byreference). Further, a molecule with an Ig domain structure similar toICAM-1 and which may function in cellular adhesion is a polio virusreceptor (Mendelsohn, C. L., et al., Cell 56:855-865 (1989)). This maybe more than coincidental, since cell adhesion and virus adhesion are inprinciple very similar. It, therefore, appears that the region of thecell adhesion molecule adopted for binding by the virus is similar tothe region adapted for binding to the cell adhesion receptor.

Binding sites for LFA-1 and HRV were determined using site-directedmutagenesis. Regions on ICAM-1 were defined by deleting domains andmaking amino acid substitutions by site-directed mutagenesis.Characterization of the binding site on ICAM-1 for LFA-1 providesinsight into the interaction between Ig and integrin superfamilymembers.

Example 37 Generation of ICAM-1 Mutants Oligonucleotide-DirectedMutagenesis

The coding region of an ICAM-1 cDNA in a 1.8 kb Sal1-Kpn1 fragment, wassubcloned into the expression vector CDMB (Seed, B. et al., Proc. Natl.Acad. Sci. (U.S.A.) 84:3365-3369 (1987)). Based on the method of Kunkel,T., (Proc. Natl. Acad. Sci. (U.S.A.) 82:488-492 (1985)) andmodifications of Staunton D. et al. (Staunton, D. E. et al., Cell52:925-933 (1988)), this construct (pCD1.8) was used to generate asingle strand uracil containing template to be used inoligonucleotide-directed mutagenesis.

Briefly, E. coli strain XS127 was transformed with pCD1.8. Singlecolonies were grown in one ml of Luria Broth (LB) medium (Difco) with 13μg/ml ampicillin and 8 pg/ml tetracycline until near saturation. 100 μlof the culture was infected with R408 helper phage (Strategene) at amultiplicity of infection (MOI) of 10, and 10 ml of LB medium withampicillin and tetracycline was added for a 16 hr culture at 37° C.Following centrifugation at 10,000 rpm for one minute, and 0.22 μmfiltration of the supernatant, the phage suspension was used to infectE. coli BW313/P3 which was then plated on LB agar (Difco) platessupplemented with ampicillin and tetracycline. Colonies were picked,grown in 1 ml LB medium with ampicillin and tetracycline to nearsaturation and infected with helper phage at MOI of 10. Culture volumewas then increased to 250 ml and the cells were cultured overnight.Single strand DNA was isolated by standard phage extraction.

Mutant oligonucleotides were phosphorylated and utilized with the pCD1.8template in a second strand synthesis reaction (Staunton, D. E. et al.,Cell 52:925-933 (1988)).

Transfection

COS cells were seeded into 10 cm tissue culture plates such that theywould be 50% confluent by 16-24 hrs. COS cells were then washed oncewith TBS and incubated for 4 hrs with 4 ml RPMI containing 10% Nu sera(Collaborative) 5 pg/ml chloroquine, 3 μg of mutant plasmid and 200μg/ml DEAE-dextran sulfate. Cells were then washed with 10% DMSO/PBSfollowed by PBS and cultured 16 hrs in culture media. Culture media wasreplaced with fresh media and at 48 hrs post transfection (OS cells weresuspended by trypsin/EDTA (Gibco) treatment and divided into 2, 10 cmplates as well as 24 well tissue culture plates for HRV binding. At 72hrs cells were harvested from 10 cm plates with 5 mM EDTA/HBSS andprocessed for adhesion to LFA-1 coated plastic and immunofluorescence.

LFA-1 and HRV binding

LFA-1 was purified from SKW-3 lysates by immunoaffinity chromatographyon TS2/4 LFA-1 mAb Sepahrose and eluted at pH 11.5 in the presence of 2mM MgCl₂ and 1% octylglucoside. LFA-1 (10 μg per 200 μl per 6-cm plate)was bound to bacteriological Petri dishes by diluting octylglucoside to0.1% in PBS (phosphate buffered saline) with 2 mM MgCl₂ and overnightincubation at 4° C. Plates were blocked with 1% BSA (bovine serumalbumin) and stored in PBS containing 2 mM MgCl₂, 0.2% BSA, 0.025%azide, and 50 μg/ml gentamycin.

⁵¹Cr-labelled COS cells in PBS containing 5% FCS (fetal calf serum), 2mM MgCl₂, 0.025% azide (buffer) were incubated with or without 5 pg/mlRR1/1 and R6.5 in LFA-1 coated microtiter plates at 25° C. for 1 hour.Non-adherent cells were removed by 3 washed with buffer. Adherent cellswere eluted by the addition of EDTA to 10 mM and γ-counted.

For HRV binding studies, COS cells were reseeded in a 24 well plate. Oneday later, the confluent monolayer was washed twice with 1 ml of RPMI1640/10 mM MgCl₂/25 mM Hepes pH 7.3 (rhinovirus-14 buffer). TransfectedCOS cells were labeled with 51Cr for binding to LFA-1 coated plastic aspreviously described (Staunton, D. E., et al., Nature 339:61-64 (1989).Immunoprecipitation and indirect immunofluorescence was performed usingICAM-1 MAb RR1/1 (Rothlein, R., et al., J. Immunol. 137:1270-1274(1986)), R6.5 (Rothlein, R., et al., J. Immunol. 141:1665-1669 (1988)),LB-1 (Clark, E. A., et al., Hum. Immunol. 16:100-113 (1986)) and CL203(Maio, M., J. Immunol. 143:181-188 (1989), all of which references areincorporated herein by reference).

[³⁵S]-Met labeled HRV14 (Sherry, B. et al., J. Virol. 57:246-257 (1986)which reference is incorporated herein by reference)), 15-25,000 cpm(approximately 107 PFU) in 100 μl of HRV-buffer was added to each well.Binding occurred in 1 hr at 35° C. in a 5% CO₂ atmosphere withhorizontal rotation (100 rpm). Unbound [³⁵S] HRV14 was aspirated, COScells were gently washed with 150 ml of HRV buffer and then solubilizedwith 1% SDS in PBS for scintillation counting.

LFA-1 and HRV-14 binding to ICAM-1 mutants was corrected for binding tomock transfected cells and was normalized for the percent of COS cellsstaining with CL203 mAb and for percent of binding obtained with wildtype:

${\% \mspace{14mu} {binding}} = \frac{\begin{matrix}\left( {\left( {{\% \mspace{14mu} {mut}\mspace{14mu} {binding}} - {\% \mspace{14mu} {mock}\mspace{14mu} {binding}}} \right)/} \right. \\{\left. {\% \mspace{14mu} {mut}\mspace{14mu} {CL}\; 203\mspace{14mu} {stain}} \right) \times 100}\end{matrix}}{\begin{matrix}{\left( {{\% \mspace{14mu} {wt}\mspace{14mu} {binding}} - {\% \mspace{14mu} {mock}\mspace{14mu} {binding}}} \right)/} \\{\% \mspace{14mu} {wt}\mspace{14mu} {CL}\; 203\mspace{14mu} {staining}}\end{matrix}}$

Binding of RR1, R6.5, and LB-2 mAb was normalized to binding of CL203mAb using Specific Linear Fluorescence Intensity (SLFI):

% CL203=(mAb SLFI)×100)/CL203 mAB SLFI

Percent of wild-type ICAM-1 expressing COS cells that bound to LFA-1varied from 11-63% (mean=33%); percent of mock-transfected COS cellsbinding varied from 0-1%. Percent of [³⁵S] methionine-labeled HRV-14which bound to COS cells expressing wild-type ICAM-1 varied from 6-43%(mean=21%); percent of mock-transfected COS cell binding varied from0-4%.

[³⁵S] HRV14 binding to ICAM-1 coated plastic was performed as describedby Staunton, D. E. et al., Cell 56:849-853 (1989), which reference isincorporated herein by reference) but with modification of the HRVbuffer as indicated. Incubation conditions were 35° C., 5% CO₂ for 1hour with rotation.

Anti-ICAM-1 antibodies such as RR1/1 (Rothlein, R. et al., J. Immunol.137:1270-1274 (1986)), R6.5 (Springer, T. A. et al., U.S. patentapplication Ser. No. 07/250,446), LB-2 (Clark, E. A. et al., In:Leukocyte Typing I (A. Bernard, et al., Eds.), Springer-Verlag pp339-346 (1984)), and CL203 (Staunton, D. E. et al., Cell 56:849-853(1989), all of which references are incorporated herein by reference)have been identified. If these antibodies are capable of inhibitingICAM-1 function, they must be capable of binding to a particular site inthe ICAM-1 molecule which is also important to the ICAM-1 function.Thus, by preparing the above-described deletion mutants of ICAM-1, anddetermining the extent to which the anti-ICAM-1 antibodies can bind tothe deletion, it is possible to determine whether the deleted domainsare important for function.

Example 38 Visualization of ICAM-1 by Electron Microscopy

The ICAM-1 molecule was examined using electron microscopy. In order tovisualize the ICAM-1 molecule for electron microscopy, a solublefragment of ICAM-1 possessing all five extracellular Ig-like domains(FIG. 4) was purified from the culture media of COS cells transfectedwith an ICAM-1 mutant construct pCDsD1-5.

ICAM-1 was prepared from COS cells in the following manner. COS cells at50% confluency were transfected by DEAE-dextran method (Kingston, R. E.,In Current Protocols in Molecular Biology, Greene Publishing Associates,pp. 9.0.1-9.9.6 (1987)) which reference is incorporated herein byreference) using approximately 0 (mock) or 4 mg of plasmid/10 cm plate.

Secreted ICAM-1 was purified from the supernatants of COS cellstransfected with pCDsD1-5 as described by Marlin and Springer (Marlin,S. D. et al., Cell 51:813-819 (1987)) with minor modifications asdiscussed below. Supernatants were harvested between day 4 and 12post-transfection (200 ng/ml sICAM-1 0.22 μfiltered and passed overRR1/1-sepharose (5 ml, 5 mg/ml) at 0.5 ml/min. The column was washed andeluted with 50 mM triethanolamine HCl, 0.15M NaCl at pH 10 and pH 12.5,respectively, and fractions were neutralized immediately.

Soluble ICAM was dialyzed into 0.2 M ammonium bicarbonate, 30% glyceroland prepared for electron microscopy by rotary shadowing (Fowler, W. E.et al., J. Molec. Biol. 134:241-249 (1979)). Alternatively, the solubleICAM was sedimented through a 15-40% glycerol gradient, in 0.2 Mammonium bicarbonate, and the ICAM fractions were used directly forrotary shadowing. The sedimentation coefficient was estimated bycomparison to standard proteins in another gradient (curve extrapolatedfrom catalase at 11.3 S, and bovine serum albumin at 4.6 S). The 3.5 Sestimated for ICAM should be accurate to within ±0.5 S. Lengthmeasurements were made from prints magnified to 250,000 X, subtracting 1nm from each end for the estimated thickness of the shell of metal(Fowler, W. E. et al., J. Molec. Biol. 134:241-249 (1979)).

ICAM molecules were analyzed by sedimenting them through a glycerolgradient, in 0.2 M ammonium bicarbonate. The ICAM molecules remainednear the top of the gradient, at a sedimentation coefficient estimatedto be about 3.5 S. For a molecular mass of 92 kD, this indicates a valueof ^(f/f)min=2.0, indicative of a highly elongated molecule (Erickson,H. P., Biophys. J. 37:96a (1982)).

Rotary shadowed ICAM molecules appeared as thin rods, which were eitherstraight, or with a single bend. Molecules with a uniform curvature orwith two bends were rarely seen, suggesting a rigid rod structure with asingle hinge point. Although the angle of the bend was somewhatvariable, in most of the obviously bent molecules the angle was close to90 degrees.

Length measurements gave a value of 16.6±0.24 nm (av.±s.d., n=25) forthe straight molecules. For the bent molecules the long arm was11.8±0.12 nm, and the short arm was 6.9±0.15 nm (n=21). The total lengthof the bent molecules, 18.7 nm, was somewhat longer than that measuredfor the straight molecules. It was possible that the population ofstraight molecules contained some in which the short art was bent towardthe viewer, eclipsing the full profile. Thus, the bent moleculesprovided a more reliable population for length measurements. The rodappeared to have a uniform diameter, on the order of 2-3 nm.

The ICAM molecule was found to contain five repeats of IgG-like domains,which have dimension 4×2.5×2 nm. The total length of the ICAM molecule,18.7 nm, indicates 3.7 nm per IgG repeat, and suggests that the domainsare aligned with their long axes at a small angle to the axis of therod. Models in which two or four of the IgG-like domains are paired withone another are too short. The bend was thus at a point about two-fifthsalong the rod, suggesting that it occurs between domains 2 and 3 orbetween domains 3 and 4, and dividing it into a short and a long arm.

Example 39 Binding Of ICAM-1 Deletion Mutants to LFA-1 and HRV

ICAM-1 is an integral membrane protein, of which the extracellulardomain is predicted to be composed of 51 g-like C-domains. To localizethe site(s) of LFA-1 and HRV contact to a particular ICAM-11 g-likedomain(s), entire domains were deleted by oligonucleotide directedmutagenesis and tested functionally following expression in COS cells(FIG. 21). In addition, the cytoplasmic domain was deleted to determineits potential contribution to adhesion.

A secreted form of ICAM-1 including domains 1 through 5 was produced bymutation of the two most carboxyl extracellular residues Y452 and E453to F and a translational stop codon respectively (pCDsD1-5). The entirecytoplasmic domain of ICAM-1 was deleted (DCyt.⁻) by transforming thecodon for the carboxyl terminal transmembrane residue Y476 to atranslational stop codon. D3 and D4 and 5 were deleted using long (48bp) mutant oligonucleotides to span distal ICAM-1 sequence such thatcodons for F185 and P284 (D3⁻) and P284 and R451 (D4⁻5⁻) would be joined(FIG. 21). The desired deletion mutations were confirmed by DNAsequencing.

Following transfection, ICAM-1 mutants possessing deletions of thecytoplasmic (Y476/* or Dcyt.⁻), third (D3⁻) and fourth and fifth (D4⁻5⁻)domains were expressed in 50-60% of COS cells at similar characteristicbroad levels (FIG. 22). Immunoprecipitation and SDS-PAGE of Dcyt⁻, D3⁻,and D4⁻5⁻ ICAM-1 from COS cells, relative to wild-type, demonstrated a3, 24 and 23 kD decrease, respectively. Wild-type ICAM-1, approximately92 kD when expressed in COS cells, has a 55 kD core protein and thuseach of the eight linked glycosylation sites may possess an average 4 kDoligosaccharide. Based on the predicted glycosylation of each domain(FIG. 21), the observed decreases in mass are reasonably consistent withthe expected decreases of 3, 19 and 27 kD, respectively.

Efficiency of expression of mutant ICAM-1 in these studies has beenexamined with a panel of 4 ICAM-1 MAb. These 4 MAb, RR1/1, R6.5, LB-2and CL203 do not inhibit binding of one another to cell surface ICAM-1as shown with biotinylated MAb, confirming previous results (Marlin, S.D., et al., Cell 51:813-819 (1987), which reference is incorporatedherein by reference). They thus bind to four distinct epitopes.Following transfection ICAM-1 deletion mutants were expressed in COScells at characteristic broad levels (FIG. 22). All MAb bound to theDcyt mutant at levels equivalent to that of wild type (FIG. 23). Bindingof CL203 was decreased upon removal of D3 and eliminated upon removal ofD4 and D5. Binding of the other three MAb was unaffected for the D45mutant and was decreased, although less so than for CL203, for the D3mutant. Thus the epitopes for RR1/1, R6.5 and LB-2 are located within D1or 2 and that of CL203 within D4 or 5. The Dcyt and D45 mutants areefficiently expressed while the D3 mutant appears expressed at aboutone-half the level of wild type.

COS cells expressing all three deletion mutants adhere specifically toplastic-bound LFA-1 (FIG. 24, closed bars). Deletion of D4 and 5 had nosignificant effect on LFA-1 binding while deletion of D3 decreased LFA-1binding to an extent comparable to its decreased expression. Thus D1 and2 are sufficient for binding to LFA-1.

Amino acid substitutions in predicted β-turns in domains 1, 2 and 3 werealso generated and functionally tested following expression in COScells. The R6.5 epitope was thus localized to the sequence E111GA indomain 2 and may also involve E39 in domain 1 whereas RR1/1 and LB-2 areboth dependent on R13 in domain 1 (Table 25). In addition, RR1/1 bindingis decreased by mutations in the sequence D71GQS. Mutations eliminatingN-linked glycosylation sites at N103 and N165 result in decreased RR1/1,R6.5 and LB-2, LFA-1 HRV binding. These mutations appear to effectprocessing such that ICAM-1 dimers are generated.

Other mutations in domain 2 or 3 did not result in altered LFA-1adhesion (FIGS. 21 and 22). The amino acids in domain 1, R13 and D60 areimportant in antibody binding (Table 25).

Binding of HRV14 to ICAM-1 domain deletion mutants demonstrates that D1and 2 is also sufficient for this interaction (FIG. 24, open bars). Thedecreased binding to the D3⁻ mutant may be for the same reason mentionedabove for LFA-1 binding. However, deletion of D4 and 5 results in aconsistent decrease in binding HRV14 which is not found for LFA-1. Thusas the binding site on ICAM-1 becomes immersed into the cellularglycocalyx by the predicted 8 nm shortening when D4 and 5 are deleted,or alternatively as it becomes less flexible, it becomes less accessibleto HRV.

The binding of LFA-1 and HRV14 to D1 and 2 and the above-reported mAbepitope localization data correlate with previous mAb blocking data.Thus the ICAM-1 sites which interacts with RR1/1, R6.5 and LB-2 arelocalized to domains 1 and 2 block both LFA-1 and HRV binding, whereasthe ICAM-1 sites which interact with CL203 are localized to domains 4and 5. CL203 blocks neither cell adhesion nor virus adhesion (Maio, M.et al., J. Immunol. 143:181-188 (1989); Staunton, D. E., et al., Cell52:925-933 (1988)), which references are incorporated herein byreference).

Thus, LFA-1 and HRV binding appears to be a function of the aminoterminal Ig-like domain of ICAM-1. FIG. 20 shows an alignment of ICAMamino terminal domains.

Example 40 ICAM-1 Amino Acid Substitution Mutants

Features of the hypothetical Ig-like domains of ICAM-1 were used toguide not only the deletion experiments described above but also aminoacid substitutions. The three amino-terminal Ig-like domains of ICAM-1are predicted to possess 7 b strands each. These strands are predictedto be arranged in two sheets, which are connected by the intradomaindisulfide bond to form a “sandwich.” The loops connecting the b strandsin immunoglobulins form the antigen-binding site, and are hypothesizedto be utilized in intermolecular contacts in other Ig superfamilymembers (Williams, A. F. et al., Ann. Rev. Immunol. 6:381-405 (1988),which reference is incorporated herein by reference). The strategyfollowed was to first introduce two to four amino acid substitutions perloop in domains 1-3. If effects were found, single substitutions werethen made. Finally, in some areas of interest substitutions wereintroduced into b stands.

Mutants of ICAM-1 were generated in the following manner.Oligonucleotide-directed mutagenesis based on the method of Kunkel(Kunkel, T. A., Proc. Natl. Acad. Sci. USA 82:488-492 (1985), withmodifications by Peterson A. and Seed B. (Nature 329:842-846 (1987)),both of which references are incorporated herein by reference) wasutilized to generate ICAM-1 deletions and amino acid substitutions.Mutations were made using a single strand uracil-containing template ofICAM-1 cDNA subcloned into the expression vector CDM8 (pCD1.8), whichwas previously described (Staunton, D. E. et al., Cell 56:849-853(1989)). Mutant ICAM-1 oligonucleotides which confer a uniquerestriction site were used to prime a second strand synthesis reaction.Following a transformation into E. coli, mutants were isolated byscreening for the unique restriction sites. In general, two or moreisolates of each mutant were tested in binding studies following COScell transfection.

The results of this experiment are summarized in Table 25. In Table 25,the notation for the mutations uses the one-letter code for thewild-type sequence followed by a slash and the one-letter code for thecorresponding mutant sequence. The position of the first amino acidwithin the sequence is indicated. Wild type residues precede the slashfollowed by the residues they were substituted for in the mutant. COScells expressing ICAM-1 mutants were tested for adherence to LFA-1coated plastic and for binding 35S met-labeled HRV14. LFA-1 and HRV14binding is normalized for percent of cells expressing mutant ICAM-1 andfor binding of wild type expressing cells. Binding is presented as meanand standard error (SE) for multiple experiments (n). Effects oftwo-fold or greater were reproducible in LFA-1, HRV and mAb bindingassays and thus considered significant (bold and underlined). Thespecific linear fluorescence intensity (SLFI) of CL203 for each mutantis normalized to that of wild type CL203 SLFI (% WT) as discussed above.The SLFI of RR1/1, R6.5 and LB-2 SLFI is normalized to the CL203 SLFI ofthe mutant (% CL203) as described above.

TABLE 25 Binding of ICAM-1 Amino Acid Substitution Mutants to LFA-1 andHRV14 LFA-1 HRV SLFI Binding Binding CL203 (% CL203) Mutation % ±SE %±SE % WT ±SE RR/1 R6.5 LB-2 D1 Q1T/KA 119  23(2)  11  4(2) 230 61(2)  94115 113 Q1/E 175  53(3) 149 57(3) 135 21(3) 154 145 136 Q1/K 97 20(2) 78 29(2) 168 17(3) 109 121 106 S3VS/AGL 18  5(3)  61 32(2) 121 21(2)  5 31  9 S3/T 149  38(3) 196 72(3) 224 32(3) 111 114 117 V4/G 64 17(3)  3013(4) 111 39(3)  47  73  58 S5/T 104  12(2) 125 38(3) 251 24(3) 107 107120 K8/E 84  6(2) 132 18(2) 111 11(2) 104 121 110 R13G/EA  2  2(4)  3 1(2) 132  4(2)  0  31  0 R13/E  1  1(3)  10  5(3) 202 34(3)  4  48  4R13/K 98 16(3) 123 13(2) 189 45(4) 133 117 121 R13/Q 78 23(3)  60  0(2)161 36(3)  73  73  47 G15/SA 120  17(3) 164 23(2) 172 44(2)  89  88  89T20CS/ACT 91 22(3) 130 36(3) 148 24(3)  86  95  86 S24/A 80  8(2)  99 7(2) 158  4(2) 125 115 125 D26QPK/ALPE 30 13(3)  13  7(3) 126 10(3)  52 89  80 Q27/L 37  6(3)  57 26(4) 33  5(4)  75  75 125 E34/A  0  0(3)  6622(4) 132 23(4) 142 150 142 K39KE/ERQ 99 25(4)  61  4(3) 84  6(3)  47 93  87 K40/A 124   4(2)  89 20(2) 146  4(2) 123 106 106 G46NN/ASI 4915(4)  9  5(2) 151 24(4) 140 107 113 N48/H 88 —  81  2(2) 164 21(2) 123 94 100 R49KV/EKL 123  20(2)  49 22(2) 233 21(2) 103  97  52 K50V/EL 29 8(2)  10 8(2) 103 22(2)  23  69  23 Y52/F 72  0(2) 174 46(3) 152 10(2) 90  95 119 Y52/FA 138  35(2) 125 33(2) 100  0(2) 141 133 117 N56V/HM121  13(2) 101 42(2) 121 21(2) 106 125 125 Q58EDS/AKDI  3  3(3)  0  0(2)98 10(3)  10  22  7 Q58/H 109  13(2)  1  1(2) 135 35(2)  93 107  93E59/K 134  50(2) 105 20(2) 130  1(2) 127 109 136 E59/Q 84 38(2)  92 —195 25 112 106 125 D60S/KL  1  1(3)  1  0(2) 105 17(2)  0  21  0 D60/K14 —  4  0(2) 89  8(2)  0  31  0 D60/N 92 33(4)  89 14(3) 127 14(3) 100138 108 D60/Q 18  6(2)  20  8(2) 80  1(2)  30  54  31 S61/I 59 18(3) 111 4(2) 140 18(5)  82 100 100 Q62PM/API 104  48(3) 182 61(2) 200 29(4)  59 81  73 M64/I 111  13(2) 107  3(2) 183 40(2)  83 111 116 Y66/T 135  —204 — 144 — 109 104 113 N68/K 101   1(2) 137 23(2) 153  8(2)  97  96 102D71GQS/NGEL  1  1(4)  21 12(3) 161 54(3)  0  48  26 D71/E 118  28(2) 140 7(2) 124  6(2)  89 100  82 D71/N 79  3(3)  62  1(2) 109 26(4)  44  94 83 Q73/H 12 10(4) 117 31(5) 139 27(5)  21  80  80 Q73/T 40 12(4) 13346(2) 218 48(2)  71  86 114 S74/A 70  6(2) 156 35(2) 129 29(2) 119 119113 T75/A 59 28(2) 119  8(2) 153 10(2)  94  94 115 K77T/ES 87 22(4)  4214(4) 151 38(3)  88  80  84 Y83/S 42  9(2)  86 64(2) 125 —  60  70  50E87/K 65 10(5)  27 10(3) 94 12(3)  64  64  79 R88V/EA 95  1(2) 152  1(2)113 14(3)  74 100 121 E90/Q 122  45(3) 157 57(2) 152 17(2)  90  92 112E90/K 34 11(2)  50 22(4) 29  5(4) 100 123 108 D2 L91/A 87  7(2) 105 — 15—  79 142 133 G101K/AN 97 55(3) 140 60(2) 142 21(3)  85  85  85 N103/K12  6(2)  13 — 91  9(2)  17  60  22 E111GGA/KAGS 103  35(3) 162 73(2)122 —  81  0  89 N118/Q 54 22(3) 110 — 139  9(3)  93  85  93 R125/E 8127(3) 157 15(2) 145 37(2) 181 133 104 E127/R 82 29(2) 131 4(2) 191 22(2)100 119 106 K128/R 109  52(3) 137 37(2) 190 35(2) 100 118 109 V136GE/GVK92 53(3) 117  1(2) 171 42(2) 220 172 138 R149RD/EEG 81 40(2) 139 56(2)159 47(2) 166 189 166 H152HGA/EEGS 85 52(2)  82 20(2) 94 — 103  94 112A115N/SV  0  0(3)  12  8(2) 60  6(2)  0  0  0 N156/E 60  4(2) 107 — 18257(2)  84  95  95 R166PQ/EPA 75  8(2)  25  8(3) 94 10(3) 100 109  64N175TSA/QTLG 26 10(4)  40  8(3) 75 16(3)  19  50  44 N175/A 67  3(3) 12946(2) 80  9(2) 162 175 150 S177/G 59  1(2)  66  6(2) 87  6(3) 136 118 90 A178/G 59  3(2) 102 — 304 —  63  69  74 D3 A189T/SI 91  3(2) 168 —138 38(2) 175 166 166 D203TQ/TAD 91 53(2) 111  8(2) 125 — 178 116  86D213GL/HGV 90 52(3)  99 13(2) 128 28(2) 231 175 107 D229QR/HLE 90 37(3)106 11(2) 210 59(2)  94  92 100 N240DS/KNA 147  29(3)  82 32(2) 142 —113 125 132 E254DE/KEK 122  33(2)  99 23(2) 180 63(2) 178 129  81N269QSQI/IQAEQ 101   9(2) 108 45(2) 95 15(3) 162 150 150

Since the epitope of mAb CL203 localizes to D4 or 5, whereas D1-D3 weresubjected to amino acid substitutions, CL203 was used to determine thelevel of expression of ICAM-1 amino acid substitution mutants intransfected COS cells. Binding of ICAM-1 mutants to LFA-1 and HRV wasnormalized with respect to mAb CL203 binding and to binding obtainedwith wild-type ICAM-1. Furthermore, since mAb RR1/1, R6.5, and LB-2 bindto distinct epitopes, a loss of two or more of these epitopes indicatesa gross disruption of structure and thus corresponding effects on LFA-1and HRV binding were discounted. Two mutations, Q27/L and E90/K, whichdemonstrate a specifically reduced level of expression as measured withCL203, were also discounted as lower expression may reflect decreasedefficiency of folding of D1 where these mutations occur.

Amino acid substitution mutants which demonstrate a decrease in thebinding of only one ICAM-1 mAb suggests that the corresponding wild-typeresidues contribute to the mAb epitope (Table 25). Decreased binding ofmAb to amino acid substitution mutants demonstrates that the epitope forRR1/1 involve the residues D71 and Q73 and sequences at D26, K39 andQ62. The epitope for R6.5 is completely and specifically disrupted by amutation in the sequence at E111 in D2. LB-2 binding is specificallyaffected by mutations in sequence at R49 in D1 and R166 in D2.

Domains 1 and 2 appear to be conformationally linked. Twelve of 53mutations in D1, and a similar proportion in D2, 4 of 18, disruptbinding of RR1/1, R 6.5, and LB.2 mAb, LFA-1, and HRV. Since theseligands bind to different sites (the mAb) or only partially overlappingregions (LFA-1 and HRV, see below), the ability of mutations widelyspread throughout both D1 and D2 to have common effects suggests thatthe conformation of D1 is dependent on the conformation of D2 and viceversa. In contrast, none of the mutations in D3 affect binding of theseligands, and none of these mutations affects binding of CL203 whichlocalizes to D4 or D5. This indicates that there is substantial contactbetween D1 and D2, but that D1 and D2 are conformationally independentof D3; i.e., there may be a hinge between D2 and D3. The most disruptivemutations involve residues R13 and D60 which would be predicted in anIg-model (see below) to come into close proximity to residues in D2.Deletion of residues in D2 (residues P95-A189) has resulted in a lack ofcell surface expression, further indicating that proper folding ofICAM-1 depends on D1 and D2 interactions.

Conformational disruption in two mutations is reflected in aberrantdisulfide bond formation. Immunoprecipitation and non-reducing SOS-PAGEof 12 D1 and D2 mutants revealed that two of them, N103/K and A155N/SV,to be ICAM-1 disulfide linked dimers. Residues N103 and N156 occur closeto C108 and C159 which are predicted to form the intra-domain disulfidebridge of D2.

Mutations with the strongest effect on LFA-1 binding localized to D1.The most dramatic mutations are E34/A which completely eliminates LFA-1binding and Q73/H, which decreases it 10-fold (Table 25). A differentsubstitution at Q73, Q73/T, demonstrated a two-fold decrease. Themutations D26QPK/ALPE and G46NN/ASI decrease LFA-1 binding 2-3 fold. Inthe second domain the mutants N118/Q, N156/E, N175/A and S177/Gspecifically decreased LFA-1 binding approximately two-fold. These fourmutants were found to affect three of the four N-linked glycosylationsites in D2; there are no N-linked glycosylation sites in D1 (FIG. 21).Thus, N-linked carbohydrate may have a small but not critical role inLFA-1 binding. The effect of these mutations may be more indirect. Oneindirect effect D2 N-linked glycosylation may have is a change in theflexibility of the hinge. None of the mutations in D3 affected LFA-1binding, in agreement with the lack of effect of deleting D3.

A number of mutations demonstrate that D1 is more important than D2 inHRV binding and that HRV and LFA-1 binding sites partially overlap.Seven mutants decrease HRV14 binding but have no effect on LFA-1binding. The two which demonstrated the greatest effect involved aminoacid substitutions in D1. Q58/H virtually eliminated HRV14 binding andQ1T/KA resulted in a ten-fold decrease. Four other mutations in D1demonstrated a specific two-fold effect on HRV binding, K39KE/ERQ,R49KV/EKL, D71/N and K77T/ES. One mutation in D2, R155PQ/EPA, resultedin a four-fold decrease in HRV14 binding. D3 mutations did not affectHRV binding.

Of the 4 D1 mutations discussed above which affect LFA-1 binding, 3affect HRV binding as well. The mutants, D26QPK/ALPE and G46NN/ASI,affected HRV14 binding ten-fold and LFA-1 binding two- to three-fold.The E34/A that totally eliminates LFA-1 binding decreases HRV binding2-fold. Four mutations in D2 that decreased LFA-1 binding had little orno effect on HRV binding.

Thus residues which were identified as critical (ten-fold or greateraffect) to LFA-1 or HRV binding demonstrated a separation in function.The mutations E34/A and Q73/H which markedly decrease LFA-1 binding havea weak or a non-detectable affect on HRV binding. Conversely, mutationshave been described above that have a profound affect on HRV binding yetdo not affect LFA-1 binding. An overlap in binding sites is, however,demonstrated by two mutations which affect both LFA-1 and HRV binding.In addition, proximity of binding sites is suggested by mutations whichare adjacent in sequence position yet affect binding of either LFA-1 orHRV (discussed further below).

Ten sequences/residues important to LFA-1 and HRV14 binding were definedin D1 in contrast to one sequence and potentially three N-linkedglycosylations in D2. Residues or sequences critical to binding wereidentified in D1, not in D2. Further, none of the substitutions in D3altered binding to LFA-1 or HRV14 confirming the results of deleting D3.Thus the primary site of LFA-1 and HRV14 contact is located in D1.

The interaction of LFA-1 and HRV14 was further compared with regard tothe requirement for divalent cations. It had previously beendemonstrated that ICAM-1 on the cell surface or bound to plastic bindscell surface or purified LFA-1 in a Mg²⁺ dependent manner (Marlin, S.D., et al., Cell 51:813-819 (1987); Staunton, D. E., et al., Nature339:61-64 (1989)). The binding of the LFA-1 expressing T lymphoma lineSKW3 and HRV to purified ICAM-1 was compared on a plastic substrate.Purified ICAM-1 bound to plastic was utilized and the LFA-1 expressingT-cell line was found to bind ICAM-1 only in the presence of Mg²⁺ (FIG.25). In contrast, the binding of HRV14 to ICAM-1 did not significantlydiffer in the presence of 10 mM Mg²⁺ or 5 mM EDTA. This was confirmedover a range of ICAM-1 densities on the substrate. The LFA-1:ICAM-1 andHRV:ICAM-1 interaction are thus distinctly different in divalent cationrequirements.

The above experiments demonstrate that the extracellular region ofICAM-1 exists as a 20 nm hinged rod. This indicates that the fivepredicted Ig-like domains are extended and unpaired, and are alignedend-to-end rather than side-by-side. ICAM-1 is thus similar in overallstructure to NCAM (Beckers, A., et al., Immunochem. 11:605-609 (1974)).The total length of extracellular ICAM-1 is 18.7 nm and therefore 3.7 nmper Ig domain. The long arm of NCAM, which comprises five IgG-likedomains, had a length of 17.6-18.7 nm, essentially identical to thetotal length of the ICAM molecule (Beckers, A., et al., Immunochem.11:605-609 (1974)).

Another striking similarity in the structure of ICAM and NCAM is thatboth molecules have a bend, typically at 90 degrees but with variationindicating flexibility. In ICAM this bend is probably between twoIgG-like domains, giving a long arm with three domains and a short armwith two. The finding that the conformation of D1 and D2 are dependentupon one another indicates that the hinge is located between D2 and D3.The sequence at the D2-D3 border demonstrates the most proline richregion in ICAM-1 (4 prolines within 10 residues). This is consistentwith Ig hinge sequences which are characteristically proline rich.Indeed, all 4 prolines in this region are spaced identically to 4prolines in the hinge region of mouse IgG3.

In NCAM there is no bend within the five IgG-like domains (these formthe apparently rigid long arm equal to the total length of ICAM);rather, the bend immediately follows the sequence of IgG-like domains.The short arm of NCAM contains two fibronectin-like domains, themembrane spanning segment and cytoplasmic domain (Beckers, A., et al.,Immunochem. 11:605-609 (1974)).

Remarkably, the cell adhesion molecule LCAM, which has no IgG-likedomains and is unrelated to ICAM or NCAM, also has a 90 degree bend(Becker, J. W., et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1088-1092(1989)).

This common feature of cell adhesion molecules would thus appear to befunctionally important to permit an extended segment of the molecule,rather than just the tip, to face and form an interface with itsreceptor. It would allow binding sites located on the distal, flexiblesegment to bind to receptors oriented at different angles and located atvarying distances with respect tot the membrane of the cell bearing theICAM-1 molecule. Furthermore, segmental flexibility provided by thehinge should increase the rate of diffusion of the binding site withinthe volume of solvent above the cell surface to which it is limited byits membrane tether, thereby enhancing the kinetics of binding toadhesion receptors or viruses and increasing the efficiency of theseinteractions.

The rod-shaped unpaired domain organization of ICAM-1 thus facilitatesadhesion by elevating binding sites to a critical distance above thecell surface. Rhinovirus binding was more sensitive than LFA-1 bindingto deleting domains 4 and 5, which is predicted to shorten ICAM-1 by 7.4nm and affect its flexibility. This may be related to 2 differencesbetween rhinovirus and LFA-1. First, the binding site on HRV is proposedto be submerged in a 2.5 nm deep cleft within a canyon which forms amoat around the five-fold axis of the virion (Rossmann, M. G., et al.,Nature 317:145-153 (1985)), while electron microscopic studies ofintegrins suggest a 10×8 nm globular binding domain supported on 18nm-long stalks above the cell surface (Carrell, N. A. et al., J. Biol.Chem. 260:1743-1749 (1985); Nermut, M. V. EMBO J. 7:4093-4099 (1988)).The cellular glycoclyx (Williams, A. F. et al., Ann. Rev. Immunol.6:381-405 (1988)) into which ICAM-1 is submerged by its shortening mayrepel the bulkier rhinovirus more than LFA-1. Second, binding ofmultiple ICAM-1 molecules to rhinovirus (Colonno, R. J. et al., Proc.Natl. Acad. Sci. (U.S.A.) 85:5449-5453 (1988)) would require closeproximity of the ICAM-1 molecules one to another, and this packing maybe hindered by shortening or loss in flexibility. LFA-1 interaction withICAM-1 also requires multivalent interactions, but the LFA-1 moleculesmay well be separated from one another, and, based on content of onealpha and beta subunit each, are predicted to have one binding siteeach.

The unpaired domain nature of ICAM-1 and the location ofsequences/residues involved in binding to D1 is consistent with ICAM-1D1 binding within the deep cleft of the proposed HRV canyon bindingsite. The interface of ICAM-1 and HRV may be envisioned in at least twodifferent models. Based upon predicted secondary structure, ICAM-1sequences were positioned in an Ig fold model (FIG. 26). Four of the sixD1 sequences which were implicated in HRV contact Q1T, D26QP, G46NN, andR49KV, locate in this model to the distal half of D1. The dimension ofthe deep cleft (3-1.2 nm wide and 2.5 nm deep) is such that slightlymore than half an Ig-like domain (4 nm long and 2.5-2 nm wide) could beinserted. The distal half of ICAM-1 D1 may therefore bind to residueswithin the cleft such that the long axis of D1 is approximatelyperpendicular to the surrounding surface of the virion. The distancebetween the boundary of each deep cleft, approximately 4 nm, is greatenough to allow an ICAM-1 to occupy all five clefts around the five-foldaxis of the virion. Other sequences implicated in HRV contact, K39KE,Q58 and R46U6PQ, may interact with HRV residues in the rim of thecanyon. Alternatively, these residues in D1 and 2 may not form bondswith HRV residues but contribute inter or intra domain bonds importantto binding conformation. A second model of ICAM-1: HRV interaction wouldbe ICAM-1 D1 contacting residues of the cleft such that the long axis ofD1 would form an acute angle with the surrounding virion surface. ThusD1 would be more parallel and horizontal with the canyon. This mayresult in blocking by steric hindrance of some sites around thefive-fold axis.

Because 3 non-crossblocking ICAM-1 mAb block both LFA-1 andrhinovirus-14 binding it was suggested previously that LFA-1 andrhinovirus-14 contact sites on ICAM-1 are in close proximity (Staunton,D. E. et al., Cell 56:849-853 (1989)). Our present studies show thebinding site for rhinovirus-14. We have modeled these sequence positionson ICAM-1 domains 1 and 2 (FIG. 26) assuming an Ig domain structure(Williams, A. F. et al., Ann. Rev. Immunol. 6:381-405 (1988)) althoughthe Ig fold may differ in some important way for Ig family members withunpaired domains. Characterization of the mutants G46NN/ASI, D26QPK/ALPEand E34/A reveals common use of ICAM-1 sequences in LFA-1 andrhinovirus-14 binding. The predicted location of contact sequences inthe Ig domain model is consistent with close proximity or overlap ofLFA-1 and rhinovirus-14 binding sites. Residues implicated in LFA-1binding, such as Q73 and G46, are proximal to residues implicated inrhinovirus-14 binding, D71 and R49. Thus rhinovirus-14 appears to haveevolved to bind to a site on ICAM-1 which overlaps with the LFA-1binding site. The two binding sites are clearly distinguished, however,by mutations at E34 and Q58 which dramatically and selectively abolishLFA-1 and rhinovirus binding, respectively. Three of the four D1sequences implicated in LFA-1 contact and 6 of the 9 sequencesimplicated in rhinovirus-14 contact locate to the membrane-distal halfof D1 in this model; however, some of the sites where mutations have themost dramatic effect localize to the proximal half. The overlap ofrhinovirus and LFA-1 binding sites in domain 1 appears to be aconsequence of the favorability of this domain as an adhesiveinteraction site as outlines above. Alternatively, ICAM-1 might be areceptor with a triggering function in antigen-presenting cells. In thisscenario, binding to domain 1 would trigger through ICAM-1a responsethat would be advantageous to rhinovirus, for example by stimulatingnasal secretions that would help spread the virus to other people. Thiswould be an example of evolutionary mimicry.

The contact site on ICAM-1 differs from that of many other integrinligands in sequence and structure. Many integrins which bindextracellular matrix proteins bind to an RGD or an RGD-like sequence intheir ligands (Ruoslahti, E., et al., Cell 44:517-518 (1986); Hynes, R.O., Cell 48:549-554 (1987)). Human ICAM-1 has no RGD sequences butseveral RGD-like sequences (Simmons, D. et al., Nature 331:624-627(1988); Staunton, D. E. et al. Cell 52:925-933 (1988)); murine ICAM-1contains an RGD sequence. However, none of these sites correspond toresidues defined by our mutagenesis studies as important in LFA-1binding to ICAM-1. Instead of a contiguous sequence like RGD, a numberof discontinuous sequences in ICAM-1 appear to be recognized. This issimilar to Ig binding to protein antigens in which residues in threenoncontiguous complementary-determining regions confer recognitionspecificity (Alzari, P. M. et al., Ann. Rev. Immunol. 6:555-580 (1988)).

ICAM-1 is able to bind another leukocyte integrin, MAC-1, which alsobinds ligands such as iC3b and fibrinogen in an RGD dependent manner.The site on ICAM-1 which binds MAC-1, however, appears to differ fromthat which binds LFA-1. Thus MAC-1 binds to an RGD-like sequence onICAM-1 which would be more consistent with its other bindingspecificities.

ICAM-1 residues which have been defined above as being important toLFA-1 binding are conserved in other ICAMs. Human ICAM-1 is 50%identical to murine ICAM-1 and 35% identical to human ICAM-2 (Staunton,D. E., et al. Nature 339:61-64 (1989)). The residues that are mostcritical to LFA-1 binding, E34 and Q73, are conserved both in mouseICAM-1 and in human ICAM-2. This is consistent with the ability of bothmouse ICAM-1 and human ICAM-2 (Staunton, D. E., et al. Nature 339:61-64(1989)) to bind to human LFA-1. One D2 N-linked glycosylation site atN156, which influences LFA-1 binding, is also conserved in ICAM-2.Several residues that are important to rhinovirus-14 binding, Q58, G46,D71, K77 and R166, are not conserved in mouse ICAM-1 or human ICAM-2which is consistent with the apparent inability of mouse cells (Colonno,R. J. et al., J. Virol. 57:7-12 (1986)) and ICAM-2 to bindrhinovirus-14.

Sequences important to LFA-1 and HRV contact also correspond to blockingmAb epitopes of RR1/1 and LB-2 whereas the R6.5 epitope does not appearto, and thus may block, binding by steric hindrance.

Binding of LFA-1 to ICAM-1 is dependent on divalent cations. Allintegrin α subunits have 3 or 4 tandem repeats of “EF hand”-likedivalent cation binding sites (Kishimoto, T. K. et al., Adv. Immunol.46:149-182 (1989)). However, these sites differ from the classicalEF-hand motif in that they lack one conserved glutamic acid whichcoordinates with divalent cations (Corbi, A. L. et al., EMBO J.6:4023-4028 (1987)). It has been hypothesized that this residue missingfrom the integrin may be replaced by a residue in the ligand, and thusthat the metal may coordinate with both the receptor and the ligand(Corbi, A. L. et al., EMBO J. 6:4023-4028 (1987)). The ICAM-1 residuemost critical to binding LFA-1, glutamic acid 34 (E34), might providethe hypothesized coordination with the divalent cation. A similarmechanism does not appear to be present in rhinovirus-14 binding toICAM-1, which has been found to be divalent cation independent. Previoussuggestions of a divalent cation requirement for rhinovirus binding(Rueckert, R. R., In: Fields Virology, Fields, B. N. et al. (eds.),Raven Press, NY, (1985) pp 705-738) appear to be based on work withminor group serotypes, which bind to a distinct receptor. Stability asopposed to binding may be influenced by cations that coordinateasparagine 141 at the 5-fold axis of rhinovirus (Rossmann, M. G., etal., Nature 317:145-153 (1985)).

ICAM-1 and CD4 are members of the Ig superfamily which demonstratestriking parallels in their function in both cellular and viraladhesion. CD4 is an adhesion receptor on T cells that binds to MHC classII molecules, and is also utilized as a receptor by HIV virus. CD4 has 4extracellular domains. Recent studies on CD4 have found that mutationsin the amino-terminal Ig-like domain have the strongest effect onbinding of MHC class II and HIV, with a lesser effect of mutations inthe second domain. The binding sites for MHC class II and HIV areoverlapping but distinct (Peterson, A. et al., Cell 54:65-72 (1988));Clayton, L. K. et al., Nature 339:548-551 (1989); Lamarre, D. et al.Science 245:743-746 (1989); Landau, N. R. et al., Nature 334:159-167(1988), all of which references are incorporated herein by reference).Some CD4 mAb epitopes appear to involve residues from both D1 and 2demonstrating close physical association of these domains (Landau, N. R.et al., Nature 334:159-167 (1988)). In all these respects, findings onthe cell adhesion and virus binding sites of ICAM-1 and CD4 are similar.

At least two different models may be envisioned for binding of ICAM-1domain 1 to the putative receptor site in tbe rhinovirus canyon. Asmentioned above, the majority (6 out of 9) of D1 sequences implicated inrhinovirus-14 contact may locate to the distal half of D1 (FIG. 26). Thereceptor binding site in the rhinovirus canyon has been implicated to bein a deep cleft, 3 nm wide at the top, 1.2 nm wide at the bottom, and2.5 nm deep (Rossmann, M. G., et al., Nature 317:145-153 (1985);Colonno, R. J. et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:5449-5453(1988); Rossmann, M. G. et al., Ann. Rev. Biochem. 58:533-573 (1989)).The dimensions of this cleft are such that slightly more than half of anIg-like domain (4 nm long and 2-2.5 nm wide) could be inserted. Thus thecontact sequences in the distal half of ICAM-1 D1 may form bonds withresidues within the cleft such that the long axis of D1 is approximatelyperpendicular to the floor of the canyon. The distance between thecenter of each deep cleft around the 5-fold axis, approximately 5 nm, isgreat enough to allow an ICAM-1 molecule to occupy all 5 clefts. Theremaining sequences implicated in rhinovirus-14 contact, K39DE, Q58 andR166PQ, that may not locate to the distal half of D1 might interact withrhinovirus-14 residues in the rim of the canyon.

A second model of ICAM-1/rhinovirus-14 interaction would be that ICAM-1D1 contacts residues of the cleft such that the long axis of D1 wouldform a more acute angle with the floor of the canyon, allowing D1 and D2to lie more lengthwise in the canyon. This may result in blocking bysteric hindrance of some neighboring rhinovirus-14 binding sites.

Thus, the present invention resolves major points of contact betweenICAM-1 and LFA-1 or HRV. Identification of ICAM-1 contact sequencesprovides additional information for the design of ICAM-1 fragments andsynthetic peptides which inhibit LFA-1 and/or HRV binding. For example,the data shows that an ICAM-1 fragment consisting of D1 alone will besufficient to inhibit both LFA-1 and HRV interaction; however, resultspresented here suggest that an even more efficient binding conformationwill contain both D1 and D2. Since discontinuous ICAM-1 sequences appearto be involved in contact, a long peptide fragment or several shorterpeptides which span multiple contact sequences may be used to competeLFA-1 and HRV interactions.

Thus, the identification here of the important binding sites within thefirst 2 domains of ICAM-1 demonstrates that soluble fragments of ICAM-1possess potential therapeutic utility in preventing rhinovirus infectionand in the treatment of inflammatory disorders and conditions (such asreperfusion injury, transplantation, etc.). Such agents may be effectivein therapeutic treatment of 50% of cases with common cold symptoms whichare caused by the major group of rhinoviruses (Sperber, S. J. et al.Antimicr. Agents Chemo. 32: 409-419 (1988)). In reperfusion injury,leukocytes migrate into and damage tissues temporarily deprived of bloodflow. Significant damage due to reperfusion injury in myocardial infarctand ischemic shock has been shown to be blocked by mAb to LFA-1 andother leukocyte integrins (Vedder, N. B. et al., J. Clin. Invest.81:939-944 (1988); Simpson, P. J. et al., J. Clin. Invest. 81:624-629(1988); which references are incorporated herein by reference).

Thus, in summary, LFA-1 (CD11a/CD18) on lymphocytes binds to ICAM-1(CD54) on other cells to promote critical cell-cell adhesion duringimmune and inflammatory responses; furthermore, the major group of humanrhinoviruses (HRV) utilized ICAM-1 as its cellular receptor. Electronmicrographs show the ICAM molecule to be a rod, about 19 nm long. Therod frequently has a 90 degree bend, giving a 12 nm long arm and a 7 nmshort arm. These dimensions suggest a model in which the 51 g-likedomains are oriented at a small angle to the rod axis, with threedomains in the long arm and two in the short arm. ICAM-1 sequencesimportant to binding LFA-1, HRV, and 4 monoclonal antibodies (mAb) wereidentified through the characterization of ICAM-1 mutants possessingdeletions of its Ig-like domains and amino acid substitutions inpredicted b turns. The amino-terminal 21 g-like domains (D1 and D2) ofICAM-1 appear to conformationally interact, and N-linked glycosylationsites in O₂ appear to be important to the structural integrity and mayhave a minor effect in LFA-1 binding. The amino-terminal Ig-like domainof ICAM-1 (D1) contains the primary site of contact for both LFA-1 andHRV. The binding sites appear overlapping but distinct; HRV binding alsodiffers from LFA-1 in the lack of divalent cation dependence. AlthoughLFA-1 is an integrin, it does not recognize a RGD or RGD-like sequencein ICAM-1. Overall, the analysis suggests that rhinoviruses mimic LFA-1in the choice of binding site ICAM-1, raising the possibility that thisis an evolutionary adaptive site.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

1. ICAM-1, or a functional derivative thereof, substantially free ofnatural contaminants. 2-6. (canceled)
 7. A recombinant DNA moleculecapable of expressing ICAM-1 or a functional derivative thereof. 8.(canceled)
 9. A method for recovering ICAM-1 in substantially pure formwhich comprises the steps: (a) solubilizing ICAM-1 from the membranes ofcells expressing ICAM-1, to form a solubilized ICAM-1 preparation, (b)introducing said solubilized ICAM-1 preparation to an affinity matrix,said matrix containing immobilized antibody capable of binding toICAM-1, (c) permitting said ICAM-1 to bind to said antibody of saidaffinity matrix, (d) removing from said matrix any compound incapable ofbinding to said antibody and (e) recovering said ICAM-1 in substantiallypure form by eluting said ICAM-1 from said matrix. 10-14. (canceled) 15.A method of identifying a non-immunoglobulin antagonist of intercellularadhesion which comprises: (a) incubating a non-immunoglobulin agentcapable of being an antagonist of intercellular adhesion with alymphocyte preparation, said lymphocyte preparation containing aplurality of cells capable of aggregating, (b) examining said lymphocytepreparation to determine whether the presence of said agent inhibits theaggregation of said cells of said lymphocyte preparation; whereininhibition of said aggregation identifies said agent as an antagonist ofintercellular adhesion.
 16. A method for treating inflammation resultingfrom a response of the specific defense system in a mammalian subjectwhich comprises providing to a subject in need of such treatment anamount of an anti-inflammatory agent sufficient to suppress saidinflammation; wherein said anti-inflammatory agent is selected from thegroup consisting of: an antibody capable of binding to ICAM-1; afragment of an antibody, said fragment being capable of binding toICAM-1; ICAM-1; a functional derivative of ICAM-1; and anon-immunoglobulin antagonist of ICAM-1. 17-44. (canceled)
 45. A methodof suppressing the metastasis of a hematopoietic tumor cell, said cellrequiring a functional member of the LFA-1 family for migration, whichmethod comprises providing to a patient in need of such treatment anamount of an anti-inflammatory agent sufficient to suppress saidmetastasis; wherein said anti-inflammatory agent being selected from thegroup consisting of: an antibody capable of binding to ICAM-1; afragment of an antibody, said fragment being capable of binding toICAM-1; ICAM-1; a functional derivative of ICAM-1; and anon-immunoglobulin antagonist of ICAM-1. 46-53. (canceled)
 54. A methodof diagnosing the presence and location of inflammation resulting from aresponse of the specific defense system in a mammalian subject suspectedof having said inflammation which comprises: (a) administering to saidsubject a composition containing a detectably labeled binding ligandcapable of identifying a cell which expresses ICAM-1, and (b) detectingsaid binding ligand. 55-68. (canceled)
 69. A pharmaceutical compositioncomprising: (a) an anti-inflammatory agent selected from the groupconsisting of: an antibody capable of binding to ICAM-1; a fragment ofan anti-body, said fragment being capable of binding to ICAM-1; ICAM-1;a functional derivative of ICAM-1; and a non-immunoglobulin antagonistof ICAM-1, and (b) at least one immunosuppressive agent selected fromthe group consisting of: dexamethasone, azathioprine and cyclosporin A.70. (canceled)